WO2019244901A1

WO2019244901A1 - Method for separating antibody, and method for testing on disease

Info

Publication number: WO2019244901A1
Application number: PCT/JP2019/024160
Authority: WO
Inventors: 陽介寺尾; 泰之秋山; 諭遠藤; 遼子渡邉
Original assignee: 東ソー株式会社
Priority date: 2018-06-20
Filing date: 2019-06-18
Publication date: 2019-12-26
Also published as: CN112334768B; US20210116445A1; JP2020118664A; EP3812759A4; EP3812759A1; JP7469584B2; CN112334768A

Abstract

The present invention addresses the problem of providing a method for detecting the presence or absence of a disease, the risk of development of a disease, the degree of progression of a disease and/or the degree of progression of aging in a subject. The problem can be solved by a method comprising the following steps (a) to (c): (a) adding a solution containing an antibody obtained from a subject to a column filled with an insoluble carrier having an Fc-binding protein immobilized thereon, thereby causing the adsorption of the antibody onto the carrier; (b) eluting the antibody adsorbed on the carrier using an eluting solution, thereby obtaining data associated with a separation pattern of the antibody; and (c) detecting the presence or absence of a disease, the risk of development of a disease, the degree of progression of a disease and/or the degree of progression of aging in the subject employing the data as a measaure.

Description

Antibody separation method and disease test method

(4) The present invention relates to a method for separating an antibody and its use. The present invention, specifically, for example, as an index the characteristics of the separation pattern when separating antibodies obtained from the subject, the presence or absence of the disease in the subject, the risk of developing the disease, the degree of disease progression, and And / or a method for detecting the degree of progress of aging.

In recent years, drugs containing antibodies (antibody drugs) have been used for the treatment of cancer and immune diseases. Antibodies used for antibody drugs are purified to high purity using column chromatography or the like after culturing cells capable of expressing the antibody (eg, Chinese hamster ovary (CHO) cells) obtained by genetic engineering techniques. Manufactured. However, recent studies have revealed that antibodies produced in such a manner are subjected to modifications such as oxidation, reduction, isomerization, and glycosylation to form aggregates of various molecules. There are concerns about the effects on drug efficacy and safety. In particular, it has been reported that the sugar chain structure bound to an antibody greatly affects the activity, kinetics, and safety of an antibody drug, and detailed analysis of the sugar chain structure is important (Non-patent Documents) 1). In diseases such as rheumatism, changes in the sugar chain structure added to antibodies in blood are known (Non-Patent Documents 2 and 3), and the disease is analyzed by analyzing the sugar chain structure added to the antibodies. Diagnosis may be possible.

ＬＣ As a method for analyzing the sugar chain structure of an antibody used for an antibody drug, LC-MS analysis including cutting out of a sugar chain (Patent Documents 1 and 2) is mainly performed. However, the analysis method involves a very complicated operation and requires a lot of time. A simpler method for analyzing the molecular structure of an antibody is analysis by chromatography. Specifically, it is possible to separate and quantify aggregates and decomposed products by separating antibodies based on molecular weight using gel filtration chromatography. In addition, the difference in charge of the antibody molecule can be separated by ion exchange chromatography. However, in the analysis by the above-mentioned chromatography, since a minute structural change of the antibody molecule such as a sugar chain structure cannot be identified, the obtained analysis result is limited.

On the other hand, it has been reported that the performance of an antibody can be measured and judged by an analysis based on the affinity between an antibody and an affinity ligand immobilized on an insoluble carrier (Patent Document 3). However, separation of antibodies due to differences in sugar chain structure, particularly separation of human-derived antibodies due to differences in sugar chain structure, has not been performed.

JP 2016-194500 A JP 2016-099304 A WO2013 / 120929

は An object of the present invention is to provide a method for separating an antibody. In one embodiment, an object of the present invention is to provide a method for detecting the presence or absence of a disease in a subject, the risk of developing the disease, the degree of progress of the disease, and / or the degree of progress of aging.

The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, it was found that antibodies can be separated based on differences in sugar chain structure by using Fc-binding proteins, and that antibodies obtained from subjects can The present inventors have found that the presence / absence of a disease, the risk of developing the disease, the degree of progression of the disease, and / or the degree of progression of aging in the subject can be detected using the characteristics of the separation pattern obtained when separating using proteins as an index. The invention has been completed.

That is, the present invention can be exemplified as follows.
[1]
The presence or absence of the disease, the risk of developing the disease, the degree of progress of the disease, and / or a method for detecting the degree of progress of aging,
The following step (c):
(C) detecting the presence or absence of a disease in the subject, the risk of developing the disease, the degree of progression of the disease, and / or the degree of progression of aging in the subject using the data relating to the antibody separation pattern as an index,
The data is characteristic of the separation pattern of the antibody,
The method wherein the data is obtained by the following steps (a) and (b):
(A) adding a solution containing an antibody obtained from the subject to a column packed with an insoluble carrier having Fc-binding protein immobilized thereon, and adsorbing the antibody to the carrier;
(B) a step of eluting the antibody adsorbed on the carrier using an eluent to obtain the data.
[2]
The method comprising the steps (a) and (b) before the step (c).
[3]
The above method, comprising a step of adding an equilibration liquid to the column before the step (a) to equilibrate the column.
[4]
The method wherein the obtaining of the data comprises obtaining a separation pattern of the antibody, and extracting the feature from the separation pattern.
[5]
The method, wherein the characteristic is peak area and / or peak height.
[6]
The method wherein the characteristic is peak area% and / or peak height%.
[7]
The method, wherein the characteristic is a characteristic of one or more peaks selected from a first peak, a second peak, and a third peak.
[8]
The method wherein the feature is a feature of a first peak.
[9]
The method wherein the step (c) comprises comparing the data with data relating to an antibody separation pattern obtained from a control subject.
[10]
The above method, wherein the disease is one or more diseases selected from cancer, autoimmune disease, infectious disease, allergy, inflammatory disease, cachexia, and age-related disease.
[11]
The above method, wherein the disease is one or more diseases selected from pancreatic cancer, stomach cancer, breast cancer, colon cancer, kidney cancer, rheumatism, Sjogren's syndrome, and pancreatitis.
[12]
The above method, wherein the Fc binding protein is any one of the following polypeptides (1) to (4):
(1) It includes the 17th to 192nd amino acid residues of the amino acid sequence of SEQ ID NO: 1, provided that at least the 176th valine is substituted with phenylalanine in the 17th to 192nd amino acid residues. A polypeptide;
(2) It includes the 17th to 192nd amino acid residues of the amino acid sequence of SEQ ID NO: 1, provided that at least the 27th valine is the glutamic acid and the 29th amino acid is the 17th to the 192nd amino acid residues. Phenylalanine to isoleucine, 35th tyrosine to asparagine, 48th glutamine to arginine, 75th phenylalanine to leucine, 92nd asparagine to serine, 117th valine to glutamic acid, 121st glutamic acid Is a polypeptide in which is substituted with glycine, phenylalanine at position 171 with serine, and valine at position 176 with phenylalanine;
(3) It includes the 17th to 192nd amino acid residues of the amino acid sequence of SEQ ID NO: 1, provided that at least the 17th to 192nd amino acid residues have at least 27th valine in glutamic acid and 29th in glutamic acid. Phenylalanine to isoleucine, 35th tyrosine to asparagine, 48th glutamine to arginine, 75th phenylalanine to leucine, 92nd asparagine to serine, 117th valine to glutamic acid, 121st glutamic acid Has been replaced by glycine and phenylalanine at position 171 by serine;
(4) A polypeptide comprising the amino acid sequence of the polypeptide of any of (1) to (3) above, provided that the amino acid sequence has 1 to 10 amino acid mutations at positions other than the above substitutions.
[13]
A composition containing two or more antibodies, wherein the composition falls under two or more of the following I to IX:
I. A value obtained by dividing the content of the antibody having G1Fa by the content of the antibody having G0F is 0.4 or less by weight;
II. A value obtained by dividing the content of the antibody having G2F by the content of the antibody having G0F is 0.2 or less by weight;
III. The content of the antibody with G2F + 2SA divided by the content of the antibody with G0F is less than or equal to 0.03 by weight;
IV. The value obtained by dividing the content of the antibody having G1Fb by the content of the antibody having G1Fa is 0.5 or more by weight;
V. A value obtained by dividing the content of the antibody having G2F by the content of the antibody having G1Fb is 0.6 or less by weight;
VI. A value obtained by dividing the content of the antibody having G2F + SA by the content of the antibody having G1Fb is 0.3 or less by weight;
VII. The content of the antibody with G2F + 2SA divided by the content of the antibody with G1Fb is less than or equal to 0.12 by weight;
VIII. The ratio of the content of the antibody having G2 + SA to the total content of the antibody is 0.2% or less by weight;
IX. The ratio of the content of the antibody having G2 + 2SA to the total content of the antibody is 0.2% or less by weight.
[14]
A composition containing two or more antibodies, wherein the composition falls under two or more of the following I to IX:
I. A value obtained by dividing the content of the antibody having G1Fa by the content of the antibody having G0F is 1.8 or more by weight;
II. A value obtained by dividing the content of the antibody having G2F by the content of the antibody having G0F is 0.6 or more by weight;
III. The content of the antibody with G2F + 2SA divided by the content of the antibody with G0F is greater than or equal to 0.06 by weight;
IV. A value obtained by dividing the content of the antibody having G1Fb by the content of the antibody having G1Fa is 0.3 or less by weight;
V. The content of the antibody with G2F divided by the content of the antibody with G1Fb is greater than or equal to 3.0 by weight;
VI. A value obtained by dividing the content of the antibody having G2F + SA by the content of the antibody having G1Fb is 0.6 or more by weight;
VII. The content of the antibody with G2F + 2SA divided by the content of the antibody with G1Fb is greater than or equal to 0.3 by weight;
VIII. The ratio of the content of the antibody having G2 + SA to the total content of the antibody is 2% or more by weight;
IX. The ratio of the content of the antibody having G2 + 2SA to the total content of the antibodies is 0.6% or more by weight.

The figure which shows the separation pattern at the time of analyzing a monoclonal antibody with the column packed with the Fc binding protein fixed gel. The figure which shows the separation pattern at the time of analyzing the gamma globulin derived from a human with the column packed with the Fc binding protein immobilized gel. The figure which shows the sugar chain structure of the antibody contained in the fraction isolate | separated and fractionated by the column packed with the Fc binding protein immobilized gel, and the monoclonal antibody. The figure which shows the sugar chain structure of the antibody contained in the fraction isolate | separated and fractionated by the column packed with the Fc binding protein immobilized gel from human-derived gamma globulin. The figure which shows the separation pattern at the time of analyzing the gamma globulin derived from the human of different age with the column packed with the Fc binding protein fixed gel. The figure which shows the plot of the 1st peak area% value at the time of analyzing the gamma globulin from a human of different age with the column packed with the Fc binding protein immobilized gel. The figure which shows the plot of the 1st peak area% value when the gamma globulin derived from a cancer patient is analyzed by the column packed with the Fc-binding protein immobilized gel. The figure which shows the plot of the 1st peak area% value at the time of analyzing the gamma globulin derived from a patient with an autoimmune disease with the column packed with the Fc binding protein immobilized gel. The figure which shows the plot of the correction | amendment 1st peak area% value when the gamma globulin derived from a cancer patient is analyzed by the column packed with the Fc binding protein immobilized gel. The plot of the corrected first peak area% value and the third peak area when analyzing gamma globulin derived from pancreatic cancer and pancreatitis patients on a column packed with an Fc-binding protein-immobilized gel and the ROC curve for pancreatic cancer and pancreatitis are shown. FIG. The figure which shows the plot of the correction | amendment 1st peak area% value when gamma globulin derived from a healthy person who smokes and does not smoke is analyzed by the column packed with the Fc-binding protein immobilized gel. The figure which shows the plot of the 1st peak height% and the 1st peak area% value, when the human-derived gamma globulin of different ages is analyzed by the FcR9_F column or the FcR9_V column packed with the Fc binding protein immobilized gel.

Hereinafter, the present invention will be described in detail.

<1> Method The present invention provides a method for separating an antibody using an Fc-binding protein. This method is also referred to as “separation method of the present invention”.

The separation method of the present invention may specifically be a method for separating an antibody, comprising the following steps (a) and (b):
(A) adding a solution containing an antibody to a column filled with an insoluble carrier having Fc-binding protein immobilized thereon, and adsorbing the antibody to the carrier;
(B) a step of eluting the antibody adsorbed on the carrier using an eluate.

Steps (a) and (b) are also referred to as “adsorption step” and “elution step”, respectively.

抗体 A separated antibody may be obtained by the separation method of the present invention. That is, one embodiment of the separation method of the present invention may be a method of producing an isolated antibody by separating the antibody using an Fc-binding protein. That is, one embodiment of the elution step may be a step of eluting the antibody adsorbed on the carrier using an eluent to obtain the eluted antibody. In other words, one embodiment of the elution step may include a step of eluting the antibody adsorbed on the carrier using an eluate, and a step of obtaining the eluted antibody. This method is also referred to as “the antibody production method of the present invention”.

The antibody production method of the present invention may specifically be a method for producing an isolated antibody, comprising the following steps (a) and (b):
(A) adding a solution containing an antibody to a column filled with an insoluble carrier having Fc-binding protein immobilized thereon, and adsorbing the antibody to the carrier;
(B) a step of eluting the antibody adsorbed on the carrier using an eluate to obtain the eluted antibody.

データ Data on the antibody separation pattern may be obtained by the separation method of the present invention. That is, one embodiment of the separation method of the present invention may be a method of producing data relating to an antibody separation pattern by separating an antibody using an Fc-binding protein. That is, one embodiment of the elution step may be a step of eluting the antibody adsorbed on the carrier using an eluate to obtain data relating to the antibody separation pattern. In other words, one embodiment of the elution step may include a step of eluting the antibody adsorbed on the carrier using an eluent, and a step of obtaining data relating to an antibody separation pattern. This method is also referred to as the “data manufacturing method of the present invention”. Data relating to the antibody separation pattern is also referred to as “separation data”. “Data measurement”, “data acquisition”, and “data production” may be used interchangeably.

The data production method of the present invention may specifically be a method for producing separation data, comprising the following steps (a) and (b):
(A) adding a solution containing an antibody to a column filled with an insoluble carrier having Fc-binding protein immobilized thereon, and adsorbing the antibody to the carrier;
(B) a step of eluting the antibody adsorbed on the carrier using an eluate to obtain separation data.

として Using the separated data as an index, the presence / absence of a disease in the subject, the risk of developing the disease, the degree of progress of the disease, and / or the degree of progress of aging can be detected. That is, the separated data may be regarded as data used as an index for detecting the presence or absence of a disease, the risk of developing the disease, the degree of progress of the disease, and / or the degree of progress of aging in the subject. Specifically, the presence / absence of a disease, the risk of developing a disease, the progress of a disease, and the like in a subject are determined by using, as an index, separation data obtained by separating an antibody obtained from the subject using an Fc-binding protein. The degree and / or the aging progress degree can be detected. That is, the present invention uses the separation data obtained by separating an antibody obtained from a subject using an Fc-binding protein as an index to determine the presence or absence of a disease, the risk of developing a disease, Provided is a method for detecting the degree of progress and / or the degree of progress of aging. This method is also referred to as “the detection method of the present invention”. The presence or absence of a disease, the risk of developing the disease, the degree of progress of the disease, and / or the degree of progress of aging are collectively referred to as “risk”. “Detection of risk”, “evaluation of risk”, and “judgment of risk” may be used synonymously.

Specifically, the detection method of the present invention may be a method for detecting the presence or absence of a disease, the risk of developing the disease, the degree of progress of the disease, and / or the degree of progress of aging, including the following step (c). :
(C) a step of detecting the presence or absence of a disease, the risk of developing the disease, the degree of progress of the disease, and / or the degree of progress of aging in the subject using the separated data as an index, wherein the data comprises the following steps: Steps obtained according to (a) and (b):
(A) adding a solution containing an antibody obtained from the subject to a column packed with an insoluble carrier having Fc-binding protein immobilized thereon, and adsorbing the antibody to the carrier;
(B) a step of eluting the antibody adsorbed on the carrier using an eluent to obtain the data.

The detection method of the present invention may include steps (a) and (b). That is, the detection method of the present invention more specifically includes the following steps (a) to (c), the presence or absence of a disease, the risk of developing the disease, the degree of progress of the disease, and / or the degree of progress of aging. The detection method may be:
(A) adding a solution containing an antibody obtained from a subject to a column filled with an insoluble carrier having Fc-binding protein immobilized thereon, and adsorbing the antibody to the carrier;
(B) a step of eluting the antibody adsorbed on the carrier using an eluate to obtain separation data;
(C) detecting the presence or absence of a disease, the risk of developing the disease, the degree of progress of the disease, and / or the degree of progress of aging in the subject using the data as an index;

Step (c) is also referred to as “detection step”.

These methods are collectively referred to as “method of the present invention”.

<Adsorption process>
The adsorption step is a step of adding a solution containing an antibody to a column packed with an insoluble carrier having Fc-binding protein immobilized thereon and adsorbing the antibody to the carrier.

“Antibody” means a molecule containing an Fc region. The antibody may be composed of an Fc region, and may contain other regions in addition to the Fc region. Examples of the Fc region include an Fc region of an immunoglobulin. The antibody may have a sugar chain added thereto. The antibody may have, for example, a sugar chain added to at least its Fc region. The antibody may be a monoclonal antibody or a polyclonal antibody. The origin of the antibody is not particularly limited. Antibodies can be from a single organism or from a combination of two or more organisms. The antibody may be, for example, a chimeric antibody, a humanized antibody, a human antibody, or a variant thereof (eg, an amino acid substitution). Antibodies include immunoglobulins. Immunoglobulins include IgG, IgM, IgA, IgD and IgE. Immunoglobulins include in particular IgG. Examples of IgG include IgG1, IgG2, IgG3, and IgG4. Examples of the antibody also include bispecific antibodies (bispecific antibodies), fusion antibodies of the Fc region with other proteins, and antibodies with artificially modified structures such as a complex of the Fc region and a drug (ADC). Can be The antibody may be, for example, an antibody drug. Antibody drugs include infliximab, an anti-TNF-α antibody, tocilizumab, an anti-IL-6 antibody, and trastuzumab, an antibody to the oncogene HER2. Antibodies can be produced by, for example, production cells such as CHO cells, Sp2 / 0 cells, NS0 cells, and hybridoma cells.

Examples of the sugar chain include the sugar chain structures described in FIGS. In particular, G0, G0F, G1, G0F + GN, G1Fa, G1Fb, G1F + GN, G2, G2F, G1F + SA, G2F + SA, G2F + 2SA, G2F + GN, G2 + SA, G2 + 2SA, S1, S2 are sugar chains that can contribute to antibody separation. No.

Human-derived antibodies usually contain antibodies having sialic acid. The content of an antibody having sialic acid in a human-derived antibody can be, for example, about 0.1 to 20% by weight based on the total content of the antibody. In human-derived antibodies, two sialic acids often bind to the sugar chain terminal. Furthermore, human-derived antibodies can usually contain bisecting GlcNAc (represented by "+ GN" in Table 5) in an amount of about 1 to 20% by weight based on the total content of the antibody. On the other hand, antibodies derived from hamsters and mice usually do not contain bisecting GlcNAc, and the number of sialic acid bonds at the sugar chain terminals is 0 to 1.

抗体 The antibody to be subjected to the adsorption step may be a mixture containing a plurality of types of antibody molecules. The antibody to be subjected to the adsorption step may be, specifically, a mixture containing a plurality of types of antibody molecules having different sugar chain structures. More specifically, the antibody subjected to the adsorption step may be a mixture containing a plurality of types of antibody molecules having different sugar chain structures added to the Fc region.

場合 When the purpose is to detect a risk, the method of the present invention may be performed using an antibody obtained from a subject.

"Subject" means a human individual whose risk is to be detected. The subject referred to here is also referred to as a “target subject” to be distinguished from a control subject described later. The subject is not particularly limited as long as an antibody sample derived therefrom can be used, that is, an antibody sample can be obtained or has already been obtained. The subject may be male or female. The subject may be an individual of any age, such as a child, a young person, a middle age, an old man, and the like. The subject may or may not be a healthy subject.

"Antibody sample" means a sample containing an antibody. Examples of antibody samples include blood samples such as blood (whole blood), diluted blood, serum, plasma, cerebrospinal fluid, umbilical cord blood, and component blood; urine, saliva, semen, feces, sputum, amniotic fluid, ascites, and other blood-derived components A fragment or a cell of a tissue such as a liver, a lung, a spleen, a kidney, a skin, a tumor, a lymph node or a cell; and an antibody separated therefrom. The antibody sample may be used in the adsorption step as it is or after appropriately subjected to a pretreatment. The pre-processing may be performed by, for example, a common method. Examples of the pretreatment include centrifugation and purification by a column. Specifically, for example, gamma globulin may be purified and used in the adsorption step. The antibody sample is used in the adsorption step in the form of a solution containing the antibody. That is, the antibody sample may be appropriately prepared in the form of a solution containing the antibody and used in the adsorption step. For example, the antibody sample as described above or a pretreated product thereof may be appropriately dissolved, suspended, dispersed, or solvent-exchanged in a liquid medium and used as a solution containing the antibody in the adsorption step. For such a liquid medium, for example, the description of an equilibration liquid described later can be applied mutatis mutandis. The liquid medium may or may not be the same as the equilibration liquid.

記載 The description of antibodies obtained from a subject can be applied mutatis mutandis to the use of other antibodies. For example, any antibody other than the antibody obtained from the subject may be used in the adsorption step in the form of a solution containing the antibody as it is, or after appropriately subjecting it to a pretreatment.

“Fc-binding protein” means a protein that has the ability to bind to the Fc region of an antibody. The Fc binding protein is not particularly limited as long as a desired antibody separation pattern can be obtained. Antibodies can be separated based on, for example, differences in affinity for Fc-binding proteins based on differences in the sugar chain structure of the antibody (eg, the sugar chain structure of the Fc region). The affinity (specifically, the sugar chain structure of the antibody) between the antibody and the Fc-binding protein can be correlated with, for example, the function of the antibody, such as its efficacy. Further, the affinity between the antibody and the Fc-binding protein (specifically, the sugar chain structure of the antibody) can be correlated with, for example, the risk in the subject. That is, the Fc-binding protein is preferably a protein that has, for example, an ability to bind to the Fc region of an antibody and can recognize a difference in the sugar chain structure of the antibody (eg, the sugar chain structure of the Fc region). Examples of the Fc binding protein include a human Fc binding protein. Examples of the human Fc-binding protein include an Fc-binding protein found in humans and variants thereof. Specific examples of the human Fc-binding protein include proteins containing the full-length or partial amino acid sequence of the extracellular region of human FcγRIIIa. Examples of the amino acid sequence of the extracellular region of human FcγRIIIa include, in the case of natural human FcγRIIIa, the region from the 17th glycine to the 192nd glutamine in the amino acid sequence shown in SEQ ID NO: 1. The partial sequence of the amino acid sequence of the extracellular region of human FcγRIIIa includes, for example, the amino acid sequence of a region capable of expressing a function of binding to at least the Fc region (for example, the Fc region of human IgG) in the extracellular region of human FcγRIIIa. Can be Examples of the human Fc-binding protein include the following polypeptides (i) and (ii).
(I) a polypeptide comprising at least the 17th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1;
(Ii) contains at least the 17th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1, and includes substitution, insertion, or deletion of one or more amino acid residues in the amino acid residues Polypeptide.

As one embodiment of (ii), the amino acid sequence includes the 17th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1 and the following (1) ) To (40), wherein the polypeptide has at least one amino acid substitution (JP-A-2015-086216).
(1) 18th methionine in SEQ ID NO: 1 is substituted with arginine (2) 27th valine in SEQ ID NO: 1 is substituted with glutamic acid (3) 29th phenylalanine in SEQ ID NO: 1 is substituted with leucine or serine (4) Leucine at position 30 in SEQ ID NO: 1 is substituted with glutamine (5) Tyrosine at position 35 in SEQ ID NO: 1 is substituted with aspartic acid, glycine, lysine, leucine, asparagine, proline, serine, threonine, or histidine (6) SEQ ID NO: Lys at position 46 of 1 is replaced with isoleucine or threonine (7) Glutamine at position 48 of SEQ ID NO: 1 is replaced with histidine or leucine (8) Alanine at position 50 of SEQ ID NO: 1 is replaced with histidine (9) SEQ ID NO: 1 No. 51 tyrosine is replaced with aspartic acid or histidine (10) The glutamic acid at position 54 of No. 1 is substituted with aspartic acid or glycine (11) The asparagine at position 56 of SEQ ID NO: 1 is substituted with threonine (12) The glutamine at position 59 of SEQ ID NO: 1 is substituted with arginine (13) Substitution of phenylalanine at position 61 with tyrosine (14) Substitution of glutamic acid at position 64 of SEQ ID NO: 1 with aspartic acid (15) Substitution of serine at position 65 of SEQ ID NO: 1 with arginine (16) Alanine at position 71 of SEQ ID NO: 1 Is substituted with aspartic acid (17) The phenylalanine at position 75 of SEQ ID NO: 1 is substituted with leucine, serine or tyrosine (18) The aspartic acid at position 77 of SEQ ID NO: 1 is substituted with asparagine (19) Position 78 of SEQ ID NO: 1 (20) Asparagine at position 82 of SEQ ID NO: 1 Is substituted with glutamic acid or valine (21) Glutamine at position 90 in SEQ ID NO: 1 is substituted with arginine (22) Asparagine at position 92 in SEQ ID NO: 1 is substituted with serine (23) Leucine at position 93 in SEQ ID NO: 1 is substituted with arginine or (24) Threonine at position 95 in SEQ ID NO: 1 is substituted with alanine or serine (25) Leucine at position 110 in SEQ ID NO: 1 is substituted with glutamine (26) Arginine at position 115 in SEQ ID NO: 1 is substituted with glutamine (27) Substitution of tryptophan at position 116 of SEQ ID NO: 1 with leucine (28) Substitution of phenylalanine at position 118 of SEQ ID NO: 1 with tyrosine (29) Substitution of lysine at position 119 of SEQ ID NO: 1 with glutamic acid (30) SEQ ID NO: Glutamic acid at position 120 of 1 is replaced with valine (31) The glutamic acid at position 121 is substituted with aspartic acid or glycine (32) The phenylalanine at position 151 of SEQ ID NO: 1 is substituted with serine or tyrosine (33) The serine at position 155 of SEQ ID NO: 1 is substituted with threonine (34) The threonine at position 163 is substituted with serine (35) The serine at position 167 of SEQ ID NO: 1 is substituted with glycine (36) The serine at position 169 of SEQ ID NO: 1 is substituted with glycine (37) The phenylalanine at position 171 of SEQ ID NO: 1 is substituted with glycine Substitution with tyrosine (38) Substitution of asparagine at position 180 of SEQ ID NO: 1 with lysine, serine, or isoleucine (39) Substitution of 185 at threonine of SEQ ID NO: 1 with serine (40) Substitution of 192 at glutamine of SEQ ID NO: 1 Replace with lysine

In another embodiment of (ii), the amino acid sequence includes the 17th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1, and the 17th to 192nd amino acid residues include And polypeptides in which at least one amino acid of (41) to (57) has been substituted (JP-A-2016-169197).
(41) The phenylalanine at position 29 in SEQ ID NO: 1 is substituted with isoleucine or leucine (42) The glutamic acid at position 39 in SEQ ID NO: 1 is substituted with glycine (43) The glutamine at position 48 in SEQ ID NO: 1 is substituted with arginine (44) The tyrosine at position 51 in SEQ ID NO: 1 is substituted with serine (45) The phenylalanine at position 61 in SEQ ID NO: 1 is substituted with tyrosine (46) The aspartic acid at position 77 in SEQ ID NO: 1 is substituted with glycine (47) The aspartic acid at position 82 is replaced with glutamic acid (48) The glutamine at position 90 of SEQ ID NO: 1 is replaced with arginine (49) The glutamine at position 112 of SEQ ID NO: 1 is replaced with leucine (50) The valine at position 117 of SEQ ID NO: 1 Is replaced with glutamic acid. (51) The lysine at position 119 in SEQ ID NO: 1 is asparagine or Substitution with glutamic acid (52) Threonine at position 140 in SEQ ID NO: 1 is substituted with isoleucine (53) Leucine at position 142 in SEQ ID NO: 1 is substituted with glutamine (54) Phenylalanine at position 171 in SEQ ID NO: 1 is substituted with serine ( 55) Leucine at position 175 of SEQ ID NO: 1 is substituted with arginine (56) Asparagine at position 180 of SEQ ID NO: 1 is substituted with serine (57) Isoleucine at position 188 of SEQ ID NO: 1 is substituted with valine

In still another embodiment of the above (ii), the amino acid sequence includes the 17th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1 and includes the 17th to 192nd amino acid residues. A polypeptide in which at least one of the following amino acids (58) to (61) is substituted (Japanese Patent Application Laid-Open No. 2016-169197).
(58) Leucine at position 66 in SEQ ID NO: 1 is substituted with histidine or arginine (59) Glycine at position 147 in SEQ ID NO: 1 is substituted with aspartic acid (60) Tyrosine at position 158 of SEQ ID NO: 1 is substituted with histidine (61) ) The valine at position 176 of SEQ ID NO: 1 is substituted with phenylalanine

In still another embodiment of the above (ii), the amino acid sequence includes the 17th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1 and includes the 17th to 192nd amino acid residues. Polypeptides in which at least one amino acid substitution among the above (1) to (61) has occurred.

The above (ii) particularly includes the following polypeptides (ii-1) to (ii-3).
(Ii-1) the amino acid sequence represented by SEQ ID NO: 1 including the 17th to 192nd amino acid residues, with the proviso that at least the 176th valine is substituted with phenylalanine in the 17th to 192nd amino acid residues. An isolated polypeptide;
(Ii-2) the amino acid sequence of SEQ ID NO: 1 including the 17th to 192nd amino acid residues, provided that at least the 17th to 192nd amino acid residues have at least 27th valine as glutamic acid, The phenylalanine at position 29 is isoleucine, the tyrosine at position 35 is asparagine, the glutamine at position 48 is arginine, the phenylalanine at position 75 is leucine, the asparagine at position 92 is serine, the valine at position 117 is glutamic acid, and the 121st is glutamic acid. A polypeptide in which glutamic acid is replaced with glycine, phenylalanine at position 171 with serine, and valine at position 176 with phenylalanine;
(Ii-3) comprising the amino acid residues from position 17 to position 192 of the amino acid sequence of SEQ ID NO: 1, with the proviso that at least the valine at position 27 is glutamic acid in the amino acid residues from position 17 to position 192; The phenylalanine at position 29 is isoleucine, the tyrosine at position 35 is asparagine, the glutamine at position 48 is arginine, the phenylalanine at position 75 is leucine, the asparagine at position 92 is serine, the valine at position 117 is glutamic acid, and the 121st is glutamic acid. A polypeptide in which glutamic acid is replaced by glycine and phenylalanine at position 171 is replaced by serine.

As long as the Fc-binding protein has a function of binding to an Fc region (for example, the Fc region of human IgG), the amino acid of the Fc-binding protein (for example, the polypeptide of (i) or (ii) above) is exemplified. The sequence may be a polypeptide containing "one or several" amino acid mutations (eg, substitutions, insertions, or deletions). “One or several” may be, for example, 1 to 50, preferably 1 to 40, more preferably 1 to 30, still more preferably 1 to 20, and particularly preferably 1 to 10. The “one or several” amino acid mutations may occur, for example, such that the amino acid substitutions of the above-exemplified Fc-binding proteins selected from the amino acid substitutions of (1) to (61) are conserved. In other words, the “one or several” amino acid mutations may occur, for example, at positions other than the amino acid substitutions of the above-mentioned Fc-binding protein selected from the amino acid substitutions (1) to (61).

As long as the Fc-binding protein has a function of binding to an Fc region (for example, the Fc region of human IgG), the amino acid of the Fc-binding protein (for example, the polypeptide of (i) or (ii) above) is exemplified. It may be a polypeptide containing an amino acid sequence having high homology to the sequence. “High homology” may mean 70% or more, 80% or more, 90% or more, or 95% or more homology. “Homology” may mean similarity or identity. “Homology” may particularly refer to identity. Amino acid sequence homology can be determined using an alignment program such as BLAST. For example, “amino acid sequence identity” may mean identity between amino acid sequences calculated using blastp, and specifically, between amino acid sequences calculated using blastp as default parameters. May be the same. The change in the amino acid sequence within the range of homology as described above may be such that, for example, the amino acid substitution of the above-described exemplified Fc-binding protein selected from the amino acid substitutions of (1) to (61) is conserved. May occur. In other words, the change in the amino acid sequence within the range of homology as described above is, for example, at a position other than the amino acid substitution of the above-described Fc-binding protein selected from the amino acid substitutions (1) to (61). May occur.

ΔFc-binding protein can be produced, for example, by expressing the gene in a host having a gene encoding the Fc-binding protein. The gene encoding the Fc binding protein can be obtained, for example, by cloning, chemical synthesis, mutagenesis, or a combination thereof. The host is not particularly limited as long as it can express the Fc-binding protein. Hosts include animal cells, insect cells, and microorganisms. Animal cells include COS cells, CHO cells, Hela cells, NIH3T3 cells, and HEK293 cells. Insect cells include Sf9 and BTI-TN-5B1-4. Microorganisms include yeasts and bacteria. Examples of the yeast include yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts of the genus Pichia such as Pichia Pastoris, and yeasts of the genus Schizosaccharomyces such as Schizosaccharomyces @ pombe. Examples of the bacteria include bacteria belonging to the genus Escherichia such as Escherichia coli. Examples of Escherichia coli include W3110 strain, JM109 strain, and BL21 (DE3) strain. Further, the Fc-binding protein can be produced, for example, by expressing a gene encoding the Fc-binding protein in a cell-free protein synthesis system.

The term “insoluble carrier” means a carrier that is insoluble in a liquid (for example, a liquid used for adsorbing or eluting an antibody, such as an equilibration solution or an eluate) passed through a column in the method of the present invention. . The insoluble carrier may have a functional group (for example, a hydroxy group) for covalently immobilizing the Fc-binding protein. Examples of the insoluble carrier include zirconia, zeolite, silica, a carrier derived from an inorganic substance such as coated silica, a cellulose, agarose, a carrier derived from a natural organic polymer such as dextran, polyacrylic acid, polystyrene, polyacrylamide, and poly. Carriers derived from synthetic organic high-molecular substances such as methacrylamide, polymethacrylate, and vinyl polymers are exemplified.

The Fc-binding protein can be immobilized on an insoluble carrier as appropriate. The Fc-binding protein can be covalently immobilized on the insoluble carrier using, for example, a functional group (for example, a hydroxy group) for covalently immobilizing the Fc-binding protein provided on the insoluble carrier. For example, when the insoluble carrier has a hydroxy group on the surface, an activating agent is used to form an activating group that can be covalently bonded to the Fc-binding protein from the hydroxy group, and the activating group and the Fc-binding protein are separated from each other. Can be covalently bonded. Specific examples of the activator for the hydroxy group include epichlorohydrin (forming an epoxy group as an activating group), 1,4-butanediol diglycidyl ether (forming an epoxy group as an activating group), and tresyl chloride ( And an activated group (to form a tresyl group) and vinyl bromide (to form a vinyl group as an activating group). Further, after converting a hydroxy group into an amino group, a carboxyl group, or the like, activation can be performed by the action of an activating agent. Specific examples of the activator for an amino group, a carboxyl group, etc. include N-succinimidyl 3-maleimidopropionate (forming a maleimide group as an activating group), 1,1′-carbonyldiimidazole (a carbonylimidazole group as an activating group) ) And halogenated acetic acid (forming a halogenated acetyl group as an activating group).

The antibody can be adsorbed to the carrier by adding a solution containing the antibody to a column packed with an insoluble carrier having Fc-binding protein immobilized thereon. The solution containing the antibody can be added to the column using, for example, a liquid sending means such as a pump. Adding a liquid to the column is also referred to as "sending the liquid to the column". The conditions for performing the adsorption step, such as the amount of the solution containing the antibody, the type of the liquid phase, the liquid phase sending speed, and the column temperature, are not particularly limited as long as the antibody is adsorbed on the carrier. The conditions for performing the adsorption step can be appropriately set according to various conditions such as the type of antibody, the type of Fc-binding protein, the type of insoluble carrier, and the scale of the column. Examples of the liquid phase include an equilibration liquid described below. For example, when the inner diameter of the column is 4.6 mm, the liquid sending speed is 0.1 mL / min to 1.5 mL / min, 0.2 mL / min to 1.0 mL / min, or 0.4 mL / min to 0 mL. .8 mL / min. The liquid sending speed may be set, for example, so as to be proportional to the square of the inner diameter of the column. The column temperature can be, for example, 0-50 ° C.

カラム Before adding the solution containing the antibody to the column, the column may be equilibrated with the equilibration solution. That is, the method of the present invention may include a step of equilibrating the column by adding an equilibration liquid to the column before the adsorption step. Equilibration solutions include aqueous buffers. Specific examples of the equilibration solution include a weakly acidic buffer having a pH of 4.0 to 6.9. The components of the buffer can be appropriately selected according to various conditions such as the pH of the buffer. The components of the buffer include phosphoric acid, acetic acid, formic acid, MES (2-morpholinoethanesulphonic acid), MOPS (3-morpholinopropanesulphonic acid), citric acid, succinic acid, glycine, and piperazine.

<Elution process>
The elution step is a step of eluting the antibody adsorbed on the carrier using an eluate.

That is, the antibody adsorbed on the carrier can be eluted by adding the eluate to the column. The conditions of the elution step, such as the type of eluate, the eluate sending format, the liquid phase sending speed, and the column temperature, are as long as the antibody is separated in a desired manner.For example, desired separation data can be obtained. As long as it is not particularly limited. The conditions for the elution step can be appropriately set according to various conditions such as the type of antibody, the type of Fc-binding protein, the type of insoluble carrier, and the scale of the column. As the eluate, one that weakens the affinity between the antibody and the Fc-binding protein can be used. The eluate includes an aqueous buffer having a lower pH than the liquid phase before elution (for example, an equilibration solution). Specific examples of the eluate include an acidic buffer having a pH of 2.5 to 4.5. For example, when the liquid phase (e.g., equilibration solution) before elution is a weakly acidic buffer having a pH of 4.0 to 6.9, the eluate may be an acidic buffer having a pH of 2.5 to 4.5. . The components of the buffer can be appropriately selected according to various conditions such as the pH of the buffer. The components of the buffer include phosphoric acid, acetic acid, formic acid, MES (2-morpholinoethanesulphonic acid), MOPS (3-morpholinopropanesulphonic acid), citric acid, succinic acid, glycine, and piperazine. The delivery format of the eluate may be, for example, a gradient or an isocratic. The delivery format of the eluate may be, in particular, a gradient. That is, elution may be performed, in particular, by increasing the proportion of eluate in the liquid phase. The gradient may be, for example, a linear gradient, a stepwise gradient, or a combination thereof. Specifically, the gradient is, for example, from 10% (v / v) to 100% (v / v) of the eluate in the liquid phase in 10 to 60 minutes, 15 to 50 minutes, or 20 to 40 minutes. May be set to increase. For example, when the inner diameter of the column is 4.6 mm, the liquid sending speed is 0.1 mL / min to 1.5 mL / min, 0.2 mL / min to 1.0 mL / min, or 0.4 mL / min to 0 mL. .8 mL / min. The liquid sending speed may be set, for example, so as to be proportional to the square of the inner diameter of the column. The column temperature can be, for example, 0-50 ° C.

分離 The separated antibody may be obtained by the elution step. The separated antibody may be obtained, for example, as an eluted fraction containing the antibody. That is, a separated antibody is obtained by collecting an eluted fraction containing the separated antibody. The eluted fraction can be collected, for example, by a conventional method. The eluted fraction can be specifically collected by, for example, an automatic fraction collector such as an autosampler. Further, the separated antibody may be recovered from the eluted fraction. The separated antibody can be recovered from the eluted fraction by a conventional method, for example. The separated antibody can be specifically recovered from the eluted fraction by, for example, a known method used for separating and purifying proteins.

(4) Separation data (ie, data relating to the separation pattern of the antibody) may be obtained by the elution step. The separation data is not particularly limited as long as it can be used as an index for detecting a risk in the subject, that is, as long as it is correlated with the risk in the subject. The separation data includes characteristics of the separation pattern of the antibody. The characteristics of the antibody separation pattern are also simply referred to as “characteristics”. That is, the step of obtaining separation data may include, for example, a step of obtaining an antibody separation pattern and a step of extracting characteristics of the antibody separation pattern (ie, extracting the characteristics from the antibody separation pattern). . The antibody separation pattern can be obtained by detecting the antibody with a detector. Examples of the detector include a UV detector and a mass detector. Examples of the antibody separation pattern include a chromatogram at the time of elution of the antibody. Features include parameters obtained from the antibody separation pattern that correlate with risk in the subject. Specifically, the characteristics include the characteristics of the elution peak (that is, the peak of the eluted antibody). Specific characteristics of the elution peak include peak area, peak elution time, peak width, number of detected peaks, and peak height. The elution peak from which features such as peak area and peak height are extracted is also referred to as “target peak”. The characteristics of the elution peak particularly include the peak area and the peak height. The antibody separation pattern may be used for feature extraction as it is or after appropriate correction such as correction of the baseline. The feature may be an absolute value or a relative value. As the relative value, the ratio or difference to the value of the other eluting peak (that is, any eluting peak other than the target peak) or the sum of the values of all the eluting peaks (that is, all the eluting peaks including the target peak) is calculated. Ratio or difference. Relative values include, in particular, ratios to the values of other eluting peaks and ratios to the sum of the values of all eluting peaks. As the other elution peak, one elution peak may be used, or two or more elution peaks may be used in combination. For example, the peak area specifically includes peak area%. "Peak area%" means the ratio (%) of the area of the target peak to the total value of the areas of all eluting peaks. Also, for example, the peak height specifically includes the peak height%. "Peak height%" means the ratio (%) of the height of the target peak to the sum of the heights of all the eluting peaks. The feature may be corrected, such as a correction based on a peak obtained from an internal standard substance or a correction based on a property of a subject. For example, the feature may be corrected based on the age of the subject. That is, for example, when the feature is affected by the age of the subject, the extracted feature may be corrected based on the age of the subject, and then used in the detection process. The characteristics corrected based on the age of the subject can be used, for example, for detecting a risk for a condition other than aging.

The target peak can be appropriately selected according to various conditions such as the type of risk. The target peak includes the first to fourth peaks. Particularly, the target peak includes a first peak, a second peak, and a third peak. More specifically, the target peak includes the first peak. The first peak can be suitably used, for example, for evaluating the risk of symptoms other than pancreatitis. The second peak can be suitably used, for example, for evaluating the risk of aging. The third peak may be suitably used, for example, for assessing risk for pancreatitis. Specifically, the third peak can be suitably used, for example, for distinguishing between pancreatitis and pancreatic cancer. As the target peak, one elution peak may be used, or two or more elution peaks may be used in combination. The “first to fourth peaks” may respectively mean the first to fourth eluting peaks after the start of elution (for example, after the start of a gradient), unless otherwise specified. The target peak may have a peak area% of 1% or more. In other words, the “first to fourth peaks” may mean, in particular, peaks having a peak area% of 1% or more, respectively, which elute first to fourth after the start of elution. The term “first peak” refers to a case where the elution step is performed by gradient elution, for example, when the pH of the liquid phase is 5.4 or less, 5.2 or less, 5.0 or less, or 4.8. It may mean the first peak to elute below. The “first peak” refers to a case where the elution step is performed by gradient elution, for example, when the pH of the liquid phase is 5.4 to 4.4, 5.2 to 4.5, or 5.5. It may mean a peak that elutes during a period that is between 0 and 4.6. The pH of the liquid phase is as follows when the pH of the liquid phase (for example, the equilibration liquid) before the start of elution is X, the pH of the eluate is Y, and the ratio of the eluate in the liquid phase is Z%. It shall be calculated by the formula (I). Further, the pH at which the peak elutes is appropriately corrected in consideration of the volume of the flow channel such as the volume of the column.
PH of liquid phase = X − ((XY) × Z / 100) (I)

<Detection process>
The detection step uses the separation data (ie, data relating to the separation pattern of the antibody) as an index to determine the risk (ie, the presence or absence of a disease, the risk of developing a disease, the degree of progress of a disease, and / or the progress of aging) in a subject. This is a step of detecting the degree.

Diseases include those affected by the activity of immune cells (eg, damaging and phagocytic effects). Immune cells include natural killer cells, monocytes, and macrophages.

Examples of the disease include cancer, autoimmune disease, infectious disease, allergy, and inflammatory disease. Any of these diseases can be diseases affected by the activity of immune cells.

Cancers include brain, breast, endometrial, cervical, ovarian, esophageal, stomach, appendix, colon, liver, gallbladder, bile duct, pancreas Cancer, adrenal gland cancer, gastrointestinal stromal tumor (GIST), mesothelioma, head and neck cancer, kidney cancer, lung cancer, osteosarcoma, Ewing sarcoma, chondrosarcoma, prostate cancer, testicular tumor, renal cells Cancer, bladder cancer, rhabdomyosarcoma, skin cancer, and anal cancer. Cancers include, in particular, pancreatic, stomach, breast, colon and kidney cancers.

Autoimmune diseases include Guillain-Barre syndrome, myasthenia gravis, multiple sclerosis, chronic gastritis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cholangitis, ulcerative colitis, Crohn's disease, primary Biliary cholangitis, autoimmune pancreatitis, Takayasu arteritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic Thrombocytopenic purpura, Basedow's disease, Hashimoto's disease, primary hypothyroidism, idiopathic Addison's disease, type 1 diabetes, chronic discoid lupus erythematosus, localized scleroderma, pemphigus, pustular psoriasis, vulgaris Psoriasis, pemphigus, gestational herpes, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo vulgaris, Sutton's acquired efferent vitiligo / Sutton's nevus, Harada's disease, autoimmune optic nerve Disease, autoimmune inner ear disorder, idiopathic Azoospermia, habitual abortion, rheumatism, systemic lupus erythematosus, antiphospholipid antibody syndrome, polymyositis, dermatomyositis, scleroderma, Sjogren's syndrome, IgG4-related disease, vasculitis syndrome, mixed connective tissue disease Can be Autoimmune diseases include, in particular, rheumatism and Sjogren's syndrome.

Infections include bacterial infections, fungal infections, parasitic protozoal infections, parasitic helminth infections, and viral infections. Bacterial infections include streptococci, Staphylococcus aureus, Staphylococcus epidermidis, Enterococci, Listeria, Meningococci, Neisseria gonorrhoeae, pathogenic Escherichia coli, Klebsiella, Proteus, Pertussis, Pseudomonas aeruginosa, Serratia, Citrobacter, Acinetobacter , Enterobacter, Mycoplasma, Clostridium, Rickettsia, Chlamydia, and other various bacterial infections; tuberculosis, nontuberculous mycobacteriosis, cholera, plague, diphtheria, dysentery, scarlet fever, anthrax, syphilis, tetanus, leprosy, Legionella pneumonia, Leptospirosis, Lyme disease, tularemia, Q fever. Fungal infections include aspergillosis, candidiasis, cryptococcosis, trichomycosis, histoplasmosis, pneumocystis pneumonia (Kalini pneumonia). Parasitic protozoan infections include amoebic dysentery, malaria, toxoplasmosis, leishmaniasis, and cryptosporidiosis. Parasitic helminth infections include echinococcosis, schistosomiasis japonica, filariasis, ascariasis, and broad-striped tapeworm infection. Viral infections include influenza, viral hepatitis, viral meningitis, viral gastroenteritis, viral conjunctivitis, acquired immunodeficiency syndrome (AIDS), adult T-cell leukemia, Ebola hemorrhagic fever, yellow fever, cold syndrome, rabies , Cytomegalovirus infection, severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), progressive multifocal leukoencephalopathy, varicella / shingles, herpes simplex, hand-foot-and-mouth disease, dengue fever, Japanese encephalitis, transmission Erythema verruca, infectious mononucleosis, smallpox, rubella, acute poliomyelitis (polio), measles, pharyngeal conjunctival fever (pool fever), Marburg hemorrhagic fever, renal symptomatic hemorrhagic fever, Lassa fever, epidemic Adenitis, West Nile fever, Herpangina, Chikungunya fever. The infection may be, for example, an opportunistic infection.

Allergies include anaphylactic shock, allergic rhinitis, conjunctivitis, bronchial asthma, urticaria, atopic dermatitis, hemolytic anemia, idiopathic thrombocytopenic purpura, drug-induced hemolytic anemia, granulocytopenia, thrombocytopenia , Goodpasture syndrome, Serum disease, Systemic lupus erythematosus (SLE), Rheumatism, Glomerulonephritis, Irritable pneumonia, Allergic bronchopulmonary aspergillosis (ABPA), Contact dermatitis, Allergic encephalitis, Transplant rejection, Tuberculosis Cavity, epithelioid granulomas.

Inflammatory diseases include diseases induced by inflammatory cytokines. Inflammatory cytokines include IL-6 and TNF-α. Specific examples of inflammatory diseases include encephalitis, osteomyelitis, meningitis, neuritis, ocular inflammation (laminaritis, scleritis, episcleritis, keratitis, chorioretinitis, retinitis, chorioretinitis) , Blepharitis, conjunctivitis, uveitis, etc.), ear inflammation (otitis externa, otitis media, otitis media, etc.), mastitis, carditis (endocarditis, myocarditis, pericarditis, etc.), blood vessels Inflammation (arteritis, phlebitis, capillary inflammation, etc.), respiratory inflammation (sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia, pleurisy, mediastinitis, etc. ), Oral inflammation (stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, siladenitis, parotitis, cheilitis, pulpitis, rhinitis, etc.), gastrointestinal inflammation (esophagitis, gastritis, gastrointestinal tract) Inflammation, enteritis, colitis, colitis, duodenitis, ileitis, appendicitis, proctitis, etc.), dermatitis, cellulitis, sweat glanditis, arthritis, dermatomyositis, myositis, synovitis, tendinitis, Panniculitis, osteomyelitis, osteomyelitis, periosteitis, nephritis, ureteritis, cystitis, ureteritis, ovitis, salpingitis, endometritis, cervicitis, vaginitis, vulvitis, orchitis, Epididymitis, prostatitis, seminal vesicle cystitis, balanitis, foreskinitis, chorioamnionitis, umbilical cord, umbilitis, hepatitis, ascending cholangitis, cholecystitis, pancreatitis, peritonitis, hypophysitis, thyroiditis , Parathyroiditis, adrenalitis, lymphangitis, lymphadenitis. Inflammatory diseases include, in particular, pancreatitis.

Specific examples of the disease include cachexia and an aging-related disease. Age-related diseases include frail (weak), sarcopenia, and locomotive syndrome. Cachexia and any of these age-related diseases can also be inflammatory diseases.

リスク The detection of the risk in the subject includes determining whether the subject has a risk or not, and determining whether the subject has a high or low risk.

The detection of the presence or absence of a disease includes determining whether the subject may or may not be currently developing the disease, and determining whether or not the subject is currently likely to have the disease. No. In addition, as detection of the risk of developing a disease, it is possible to determine whether or not a subject has the possibility of developing the disease in the future or if there is a possibility of becoming more severe, or that the subject will develop the disease in the future It is possible to determine whether sex or the likelihood of the onset of the disease when it develops is high or low. The detection of the degree of progress of the disease includes determination of whether the current degree of progress of the disease (for example, severity) in the subject is large or small. In addition, the detection of the degree of progress of aging includes determination of whether the current degree of progress of aging (for example, severity) in the subject is large or small.

That is, “there is a risk in the subject” means, for example, that the subject may currently develop the disease, that the subject may develop the disease in the future, and / or Alternatively, it may mean that if the subject develops the disease in the future, the disease may become severe. "No risk in the subject" means, for example, that the subject is not likely to develop the disease, that the subject is not likely to develop the disease in the future, and / or It may mean that there is no possibility that the examiner will become severe if the disease develops in the future. "High risk in the subject" means, for example, that the subject is likely to develop the disease at present, that the subject is likely to develop the disease in the future, This means that if the disease develops in the future, it is more likely to be severe, that the subject has a greater progression of the current disease, and / or that the subject has a greater progression of the current aging. May be. "Low risk in the subject" means, for example, that the subject is less likely to develop the disease at present, that the subject is less likely to develop the disease in the future, It means that if the disease develops in the future, it is unlikely to be severe, that the subject has a small progression of the current disease, and / or that the subject has a small progression of the current aging. May be.

The detection step can be performed using, for example, the level of the value of the separated data (that is, whether the value of the separated data is high or low) as an index. The level of the value of the separated data can be determined, for example, by comparing the value of the separated data with a predetermined threshold. In other words, the detecting step may include, for example, comparing the value of the separated data with the threshold value. That is, “the value of the separated data is high” may mean that, for example, the value of the separated data is high based on the threshold. "The value of the separated data is higher than the threshold value" means, for example, that the value of the separated data is greater than or equal to the threshold value, that the value of the separated data is greater than the threshold value, or that the value of the separated data is statistically higher than the threshold value. May mean significantly higher. "The value of the separated data is higher based on the threshold value" specifically means, for example, that the value of the separated data is 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.05 times or more of the threshold value. Times or more, 1.07 times or more, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.5 times or more, 1.7 times or more, 2 times or more, 2.5 times or more, or It may mean three times or more. Further, “the value of the separated data is low” may mean, for example, that the value of the separated data is low based on a threshold. “The value of the separated data is lower than the threshold value” means, for example, that the value of the separated data is less than or equal to the threshold value, that the value of the separated data is less than the threshold value, or that the value of the separated data is statistically lower than the threshold value. May mean significantly lower. "The value of the separated data is low with reference to the threshold value" specifically means, for example, that the value of the separated data is 0.99 times or less, 0.98 times or less, 0.97 times or less, 0.95 times or less of the threshold value. Times or less, 0.93 times or less, 0.9 times or less, 0.85 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less Or 0.3 times or less.

The value of the separation data may be classified into a risk range based on, for example, a threshold. For example, the value of the separated data may be classified into a non-risk range based on a threshold. Specifically, the value of the separated data may be classified into a dangerous range and a non-critical range based on, for example, a threshold. The “risk range” may mean a range in which there is a high possibility that the subject has a risk with respect to the value of the separated data. The “non-risk range” may mean a range in which there is a high possibility that the subject has no risk with respect to the value of the separated data. That is, if the value of the separated data is within the risk range, it may be determined that the subject has a risk or has a high risk. On the other hand, if the value of the separated data is in the non-risk range, the subject may be determined to have no risk or low risk.

For example, if the value of the peak area (eg, peak area%) and / or peak height (eg, peak height%) of the first peak and / or the second peak is high, there is a risk in the subject. Alternatively, it may be determined that the risk is high. Specifically, for example, when the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the first peak and / or the second peak is high with respect to the threshold value, The subject may be determined to be at risk or high risk. In this case, the range considered to be high based on the threshold value may be the dangerous range. More specifically, for example, the first peak area% is 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more , 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, or 30% or more, there is a risk or a high risk in the subject May be determined. Further, it is determined that the higher the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the first peak and / or the second peak, the higher the risk in the subject. May be.

For example, if the value of the peak area (eg, peak area%) and / or peak height (eg, peak height%) of the first peak and / or the second peak is low, there is no risk in the subject. Alternatively, it may be determined that the risk is low. Specifically, for example, when the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the first peak and / or the second peak is low with respect to the threshold, The subject may be determined to have no or low risk. In this case, the range considered to be low based on the threshold may be the non-risk range. More specifically, for example, the first peak area% is 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less. No or low risk in the subject when ≤16%, ≤15%, ≤14%, ≤13%, ≤12%, ≤11%, ≤10%, or ≤9% May be determined. In addition, it is determined that the lower the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the first peak and / or the second peak, the lower the risk in the subject. May be.

For example, when the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the third peak is low, it is determined that the subject is at risk or has a high risk. You may. Specifically, for example, when the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the third peak is low with respect to the threshold, the risk is low in the subject. It may be determined that there is or that the risk is high. In this case, a range considered to be low with respect to the threshold value may be a dangerous range. In addition, the lower the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the third peak, the higher the risk may be determined in the subject.

For example, when the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the third peak is high, it is determined that there is no risk or low risk in the subject. You may. Specifically, for example, when the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the third peak is high with respect to the threshold value, the risk is low in the subject. It may be determined that there is no or low risk. In this case, the range considered to be high based on the threshold value may be the non-risk range. In addition, the risk may be determined to be lower in the subject as the value of the peak area (for example, peak area%) and / or the peak height (for example, peak height%) of the third peak is higher.

場合 When using the separated data with the first peak as the target peak as an index, the risk may be selected, for example, from the risk of symptoms other than pancreatitis. When the separation data having the second peak as the target peak is used as an index, the risk may be selected from, for example, an aging risk. When the separation data with the third peak as the target peak is used as an index, the risk may be selected from, for example, the risk of pancreatitis.

Note that “determining that a subject is at risk, not, high, or low when a certain separated data meets a certain criterion (for example, low or high, or within a certain range)” It means that the subject is determined to be at risk, no, high, or low at least in the range that satisfies the criterion, and does not require that the risk be determined in the subject in the range that does not meet the criterion . However, in one aspect, a determination is made that a subject is at risk, absent, high, or low if certain separation data meets certain criteria (eg, low or high, or in a range). If "yes", the subject may be determined as having no, certain, low, or high risk within a range that does not satisfy the criterion, respectively.

The threshold value can be appropriately set by those skilled in the art according to various conditions such as the type of separated data and a desired determination accuracy. The threshold may be set, for example, for each symptom to be determined such as a disease or aging. The means for determining the threshold is not particularly limited. The threshold value can be determined, for example, according to a known method used for data analysis for dividing a population into two groups.

The threshold value can be determined based on, for example, the value of separation data of an antibody sample obtained from a control subject. The separation data of the antibody sample obtained from the control subject is also referred to as “control separation data”. The control separation data may be used in the detection step by being used in determining the threshold. The control separation data may be used for comparison with the separation data, specifically, by being used for determining a threshold value. In other words, the detecting step may include, for example, comparing the separated data with the control separated data.

Control subjects include positive controls and negative controls. "Positive control" may mean a subject that can be determined to be at risk or high. A “negative control” may mean a subject that can be determined to be at no risk or low. Examples of the positive control include individuals suffering from or having suffered from the above-mentioned diseases (particularly, the same diseases as those for which a risk is to be detected), individuals who have aged, Individuals having a combination property are included. As a negative control, an individual who does not suffer from or has not suffered from the disease exemplified above (particularly, the same disease as the disease whose risk is to be detected), an individual who has not experienced aging, Individuals having the properties of these combinations are included. The threshold may be determined based solely on the value of the separated data measured for the positive control, may be determined based solely on the value of the separated data measured for the negative control, and may be determined based on both the positive and negative controls. May be determined based on the value of the separation data measured for. The threshold may usually be determined based on the value of the separated data measured for both positive and negative controls. The number of the positive control and the negative control is not particularly limited as long as a threshold value at which the risk can be determined with desired accuracy is obtained. The number of positive and negative controls may be one, two or more, respectively. Each of the number of the positive control and the number of the negative control may usually be plural. The number of positive and negative controls may be, for example, 5 or more, 10 or more, 20 or more, or 50 or more, respectively. The number of positive and negative controls may be, for example, up to 10,000, up to 1000, or up to 100, respectively.

When determining the threshold based on only the value of the separated data measured for the positive control, for example, a value selected from the range from the upper limit to the lower limit of the value of the separated data measured in multiple individuals of the positive control, For example, an average value may be set as the threshold. Further, for example, in the distribution of the values of the separated data measured by a plurality of individuals of the positive control, the threshold value may be determined so that a predetermined ratio of the positive control is included in the risk range. The predetermined ratio may be, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 100%.

When determining the threshold based on only the value of the separated data measured for the negative control, for example, a value selected from the range from the upper limit to the lower limit of the value of the separated data measured in multiple individuals of the negative control, For example, an average value may be set as the threshold. Further, for example, in the distribution of the values of the separated data measured in a plurality of negative control individuals, the threshold value may be determined so that a predetermined ratio of the negative control is included in the non-risk range. The predetermined ratio may be, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 100%.

When determining the threshold based on both the value of the separated data measured for the positive control and the value of the separated data measured for the negative control, for example, a predetermined percentage of the positive control is included in the risk range, and , The threshold may be determined such that a predetermined percentage of the negative controls fall within the non-risk range. It is preferable that the ratio of the positive control included in the risk range and the ratio of the negative control included in the non-risk range are both higher. These proportions may be, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 100%, respectively. If it is difficult to increase both of these ratios, for example, according to various conditions such as the purpose of using the determination result by the method of the present invention, a threshold is set so that either ratio is preferentially increased. You may. For example, in order to reduce the false negative rate, a threshold value may be set so that the proportion of positive controls included in the risk range is preferentially increased.

The determination of the threshold may be performed using software, for example. For example, a statistical analysis software may be used to determine a threshold value at which a negative control and a positive control can be statistically most appropriately discriminated. Examples of such software include statistical analysis software such as “R”.

対照 The control subject also includes the target subject itself. That is, for example, the risk in the subject may be determined using the fluctuation of the separation data in the subject as an index. “The value of the separated data is high” may include a case where the value of the separated data increases. “The value of the separated data has increased” may specifically mean that the value of the separated data has increased as compared with the past value. In addition, "low value of separated data" may include a case where the value of separated data is reduced. “The value of the separated data has decreased” may specifically mean that the value of the separated data has decreased as compared with the past value. That is, the threshold value may be a past value. “Past value” means a value of separation data of an antibody sample obtained from a target subject at a certain point in the past. The target subject at some point in the past may be, for example, a positive control or a negative control.

(4) When the change in the separation data in the subject is used as an index, the increase or decrease in the risk in the subject may be determined. “At risk or high” may include an increased risk. “The risk has increased” may specifically mean that the risk has increased compared to a certain point in the past. Further, “no or low risk” may include a case where the risk is reduced. “Reduced risk” may specifically mean that the risk has decreased compared to a certain point in the past.

Note that “obtaining certain separated data and using it as an index of risk detection” is not limited to obtaining the value of the separated data itself and using it as an index of risk detection, but reflects the value of the separated data. Obtaining another value and using it as a detection index is also included. For example, when the eluting peak is composed of the first to fourth peaks, the expression “obtain the peak area% of the first peak and use it as an index for risk detection” means obtaining the value of the peak area% of the first peak itself. It is not limited to the case where the index is used as a risk detection index, and other values that reflect the value of the peak area% of the first peak, such as the total value of the peak area% of the second to fourth peaks, are obtained. Is included. In any case, numerical values such as measured values and threshold values used for detecting a risk are appropriately corrected and used according to the type of separated data. For example, when the eluting peak is composed of the first to fourth peaks, if the value of the peak area% of the first peak is X and the total value of the peak area% of the second to fourth peaks is Y, “X = 100% −Y ”holds. Therefore, when the sum of the peak area% of the second to fourth peaks (ie, “Y”) is used as the detection index instead of the value of the peak area% of the first peak itself (ie, “X”), “X satisfies a certain criterion (for example, low or high or in a certain range)” should be read as “the correction value of Y (ie,“ 100% −Y ”) satisfies the criterion”. And

The result of the risk detection may be used as an index for determining whether to perform a measure for reducing the risk to the subject (hereinafter, also referred to as “risk reduction measure”). In other words, by performing the detection method of the present invention, an index for determining whether to perform the risk reduction treatment on the subject can be obtained. That is, for example, when it is determined that the subject has a risk or is high by the detection method of the present invention, it may be determined that the subject is to be subjected to the risk reduction treatment. The detection method of the present invention may be used, for example, alone or in combination with other means, as an index for determining whether to perform a risk reduction treatment on a subject. For example, for a symptom determined to be at risk or high in a subject by the detection method of the present invention, a definitive diagnosis is performed by other means, and then it is determined that a risk reduction treatment is performed on the subject. May be. The risk mitigation action may be a medical action or a non-medical action. Examples of the risk reduction treatment include prevention and treatment of the diseases and aging exemplified above. That is, the present invention may provide a method for preventing or treating a symptom such as a disease or aging. The method of prevention or treatment includes, for example, a step of performing the detection method of the present invention, and a method of preventing or treating a subject when the risk is determined or high in the subject according to the detection method of the present invention. It may be a method for preventing or treating a symptom such as a disease or aging, which includes a step of performing a treatment. Specifically, prevention or treatment may be performed for a symptom determined to be at risk or high in a subject by the detection method of the present invention. Prevention or treatment can be performed by, for example, general means (for example, medication or surgery) for each symptom.

Specific examples are described below.

According to the method of the present invention, for example, it is possible to obtain a separation data by separating the antibody of the subject based on a sugar chain structure characteristic of a risk for a symptom such as a specific disease or aging. By comparing with such separated data, such a risk can be easily detected. Also, by obtaining separated data before and / or after any treatment (medication, surgery, etc.) for the disease and comparing it with the reference separated data, monitoring of the treatment progress of the disease and determination of a policy for the treatment can be performed. Becomes possible. The reference separation data includes separation data of a control subject. As the reference separated data, specifically, separated data of the same subject at another time point (for example, at a normal time or at the time of developing the same disease), separated data of a healthy subject, and occurrence of the same disease Separation data of different patients, and separation data serving as a reference when dividing into two groups or more groups in which a difference in response to the treatment is obtained.

For example, it is possible to evaluate a risk such as a risk of having a disease or a risk of developing a disease by using separated data of a healthy person (model) as model separated data and comparing the separated data of a subject with the model separated data. It becomes. Furthermore, by separating the fraction of the peak that is the cause of the difference between the separation data of the model and the subject, the difference in the sugar chain pattern of the antibody between the healthy subject (model) and the subject can be extracted. In addition, it is also possible to monitor the disease of the patient by using the separated data at a certain point of the patient as model separated data and comparing the separated data of the same patient at another point of time with the model separated data. When comparing two different specimen groups, it is possible to evaluate whether the difference between the separated data obtained from the two different specimen groups is a significant difference using a statistical probability (P value). It is said that the smaller the P value is, the more significant the evaluation result is. When the P value is less than the significance level, it can be said that the evaluation result has a statistically significant difference. The significance level is generally 5%.

特徴 Features of the sugar chain structure related to a specific disease include the presence or absence of addition of sialic acid, and differences in the amount of sialic acid added. In particular, in rheumatic patients, it is known that the amount of sialic acid and galactose added to the antibody decreases, and the presence or absence and content of sialic acid or galactose, It is possible to easily evaluate the difference from a person. Further, according to the method of the present invention, antibodies can be separated as a whole of sugar chain structures based on the function of Fc binding without specifying individual sugar chain structures. That is, according to the method of the present invention, a feature can be extracted as separated data based on the function of Fc binding as a whole sugar chain structure, which is not clarified by the known correlation between the amount of sialic acid and galactose and a disease. Therefore, it is possible to accurately evaluate the risk such as the presence or absence of the disease and the risk of developing the disease.

The type of sugar chain that binds to IgG is partially controlled by B cells that produce antibodies, and according to Non-Patent Document (Mol. {Cell.} Proteome., 10, M110.004655 (2011)), Since it has been reported that the sugar chain modification changes, the secretion of the cytokine can also be evaluated based on the pattern of the sugar chain binding to IgG. Cytokines are substances released from cells and are associated with various diseases. For example, it is known that the secretion of inflammatory cytokines such as IL-6 and TNF-α increases with aging, which leads to a decrease in exercise and cognitive functions such as frail (weakness) and sarcopenia. According to the method of the present invention, It is also possible to evaluate the risk of developing these age-related diseases. In addition, since the inflammatory cytokines released in association with malignant tumors cause a debilitating state (cachexia) such as weight loss of cancer patients, the method of the present invention is also useful in evaluating cachexia. It is a useful method.

The strength of the affinity of the antibody for the Fc-binding protein according to the present invention affects the activity of natural killer cells and immune cells such as monocytes and macrophages that have damage or phagocytosis on the binding substance bound by the antibody. Therefore, by detecting the difference in the affinity, it becomes possible to evaluate the risk of developing a disease affected by natural killer cells, monocytes, or macrophages, or affected by phagocytosis. Such diseases include cancer, autoimmune diseases, infectious diseases, allergies, and inflammatory diseases. Infections include opportunistic infections. Opportunistic infections are infections caused by pathogens that do not cause infection in a healthy state. Pathogens include viruses, bacteria, fungi, protozoa. In addition, the effect of the antibody acquired by vaccination or disease on activation of immune cells can be evaluated from the strength of the affinity of the antibody for the Fc-binding protein. In addition to measuring the amount of the obtained antibody in the blood, the risk of developing an infectious disease can also be accurately predicted by measuring the affinity of the antibody for the Fc-binding protein.

<2> Antibody mixture The present invention provides an antibody mixture having a specific composition. This mixture is also referred to as the antibody mixture of the present invention. The antibody mixture of the present invention can be produced, for example, by the antibody producing method of the present invention. The antibody mixture of the present invention can be produced, for example, as an eluted fraction containing the antibody.

The antibody mixture of the present invention is a composition containing two or more antibodies, and is at least two of the following I to IX (for example, 2, 3, 4, 5, 6, 7, 8) , Or 9):
I. A value obtained by dividing the content of the antibody having G1Fa by the content of the antibody having G0F is 0.4 or less by weight;
II. A value obtained by dividing the content of the antibody having G2F by the content of the antibody having G0F is 0.2 or less by weight;
III. The content of the antibody with G2F + 2SA divided by the content of the antibody with G0F is less than or equal to 0.03 by weight;
IV. The value obtained by dividing the content of the antibody having G1Fb by the content of the antibody having G1Fa is 0.5 or more by weight;
V. A value obtained by dividing the content of the antibody having G2F by the content of the antibody having G1Fb is 0.6 or less by weight;
VI. A value obtained by dividing the content of the antibody having G2F + SA by the content of the antibody having G1Fb is 0.3 or less by weight;
VII. The content of the antibody with G2F + 2SA divided by the content of the antibody with G1Fb is less than or equal to 0.12 by weight;
VIII. The ratio of the content of the antibody having G2 + SA to the total content of the antibody is 0.2% or less by weight;
IX. The ratio of the content of the antibody having G2 + 2SA to the total content of the antibody is 0.2% or less by weight.

The antibody mixture of the present invention is a composition containing two or more antibodies, and is at least two of the following I to IX (for example, 2, 3, 4, 5, 6, 7, 8) , Or 9):
I. A value obtained by dividing the content of the antibody having G1Fa by the content of the antibody having G0F is 1.8 or more by weight;
II. A value obtained by dividing the content of the antibody having G2F by the content of the antibody having G0F is 0.6 or more by weight;
III. The content of the antibody with G2F + 2SA divided by the content of the antibody with G0F is greater than or equal to 0.06 by weight;
IV. A value obtained by dividing the content of the antibody having G1Fb by the content of the antibody having G1Fa is 0.3 or less by weight;
V. The content of the antibody with G2F divided by the content of the antibody with G1Fb is greater than or equal to 3.0 by weight;
VI. A value obtained by dividing the content of the antibody having G2F + SA by the content of the antibody having G1Fb is 0.6 or more by weight;
VII. The content of the antibody with G2F + 2SA divided by the content of the antibody with G1Fb is greater than or equal to 0.3 by weight;
VIII. The ratio of the content of the antibody having G2 + SA to the total content of the antibody is 2% or more by weight;
IX. The ratio of the content of the antibody having G2 + 2SA to the total content of the antibodies is 0.6% or more by weight.

表記 The description of the sugar chain structure in the above items is as described in FIG. 3 and FIG.

用途 The use of the antibody mixture of the present invention is not particularly limited. The antibody mixture of the present invention can be used, for example, for diagnostic applications. Diagnostic uses include detection of risks (ie, the presence or absence of a disease, the risk of developing a disease, the degree of progression of a disease, and / or the degree of progression of aging) in a subject. That is, when the antibody mixture of the present invention is obtained by separating the antibody obtained from the subject by the separation method of the present invention or the like, the present invention relates to the antibody separation pattern when the antibody mixture of the present invention is obtained. The risk in the subject can be detected using the data as an index. Further, the risk in the subject may be detected using the pattern of the sugar chain structure in the antibody mixture of the present invention as an index.

Hereinafter, the present invention will be described more specifically with reference to non-limiting examples.

Preparation of Fc-Binding Protein-Immobilized Gel Example 1 Preparation of Val176 amino acid substitution of FcR9 For FcR9 (SEQ ID NO: 2) prepared by the method described in WO2015 / 199154, the 176th valine of the Fc-binding protein (SEQ ID NO: 2) An Fc-binding protein was prepared in which the 176th Val in the amino acid number of SEQ ID NO: 1 was substituted with phenylalanine. Specifically, the plasmid pET-FcR9 (WO2015 / 199154) containing a polynucleotide encoding FcR9 (SEQ ID NO: 3) was subjected to amino acid substitution using PCR, and FcR9 obtained by substituting Val176 for phenylalanine in FcR9. A binding protein was made.

In addition, FcR9 (SEQ ID NO: 2) is an Fc-binding protein containing a wild-type FcγRIII extracellular region shown in SEQ ID NO: 4, in which Val at position 43 is replaced with Glu (corresponding to position 27 in SEQ ID NO: 1) and Phe at position 45 To Ile (corresponding to position 29 in SEQ ID NO: 1), Tyr at position 51 to Asn (corresponding to position 35 in SEQ ID NO: 1), Gln at position 64 to Arg (corresponding to position 48 in SEQ ID NO: 1), The 91st Phe is Leu (corresponding to 75th in SEQ ID NO: 1), the 108th Asn is Ser (corresponding to 92nd in SEQ ID NO: 1), and the 133rd Val is Glu (117th in SEQ ID NO: 1) 137th Glu as Gly (corresponding to 121st in SEQ ID NO: 1) and 187th Phe as Ser (corresponding to 171th in SEQ ID NO: 1). ) Is an amino acid substituted Fc binding proteins.

方法 Hereinafter, the method for producing each Fc-binding protein will be described in detail.

(1) FcR9 (SEQ ID NO: 2) prepared by the method described in WO2015 / 199154 in order to replace valine at position 176 of the Fc binding protein (Val at position 176 in the amino acid number of SEQ ID NO: 1) with phenylalanine ) Using the plasmid pET-FcR9 (described in WO2015 / 199154) containing a polynucleotide (SEQ ID NO: 3) encoding SEQ ID NO: 5 (5′-TAATACGACTCACTATAGGGG-3 ′) and SEQ ID NO: 6 (5′-CATTTTTGCTGCCGAACAGCCCCACGCAGGG-). After preparing a reaction solution similar to the composition shown in Table 1 using an oligo primer having the sequence described in 3 ′), the reaction solution is heat-treated at 95 ° C. for 2 minutes, and the first step is performed at 95 ° C. for 30 seconds. A second step at 30 ° C. for 30 seconds, 30 cycles no rows react with one cycle of the third step for 90 seconds at 2 ° C., and PCR was carried out by a heat treatment and finally with 72 ° C. 7 minutes. The obtained PCR product was designated as V176p1.

(2) Plasmid pET-FcR9 (described in WO2015 / 199154) containing a polynucleotide (SEQ ID NO: 3) encoding FcR9 (SEQ ID NO: 2) prepared by the method described in WO2015 / 199154 as a template, and SEQ ID NO: 7 (5′-TATGCTAGTTATTGCTCAG-3 ′) and a reaction solution similar to the composition shown in Table 1 was prepared using an oligo primer having the sequence described in SEQ ID NO: 8 (5′-cctgccgttgggctgTTCGGCAGCAAAAATG-3 ′). A heat treatment is performed at 95 ° C. for 2 minutes, and a reaction is performed for 30 cycles in which a first step at 95 ° C. for 30 seconds, a second step at 50 ° C. for 30 seconds, and a third step at 72 ° C. for 90 seconds as one cycle. PCR was performed by heat treatment at 72 ° C. for 7 minutes. The obtained PCR product was designated as V176p2.

(3) The two PCR products (V176p1 and V176p2) obtained in (1) and (2) were mixed to prepare a reaction solution having the composition shown in Table 2. After the heat treatment of the reaction solution at 98 ° C. for 5 minutes, 5 cycles of a reaction including a first step at 98 ° C. for 10 seconds, a second step at 55 ° C. for 5 seconds, and a third step at 72 ° C. for 1 minute as one cycle. Performed PCR was performed to obtain a PCR product V176p in which V176p1 and V176p2 were ligated.

(4) PCR was performed using the PCR product V176p obtained in (3) as a template and oligonucleotides having the sequences of SEQ ID NOS: 5 and 7 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 3, the reaction solution was heat-treated at 98 ° C. for 5 minutes, a first step at 98 ° C. for 10 seconds, a second step at 55 ° C. for 5 seconds, and a reaction at 72 ° C. The reaction was performed for 30 cycles in which the 1st step was defined as one cycle. As a result, a polynucleotide encoding the Fc-binding protein in which the 176th amino acid of the Fc-binding protein (FcR9) was substituted with phenylalanine was obtained. The obtained polynucleotide was designated as V176p3.

(5) After purifying the polynucleotide obtained in (4), the polynucleotide is digested with restriction enzymes NcoI and HindIII, and ligated to an expression vector pETMalE (JP-A-2011-206046) which has been digested with restriction enzymes NcoI and HindIII in advance. Using this, Escherichia coli BL21 (DE3) strain (manufactured by Nippon Gene) was transformed.

(6) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered cells (transformants).

(7) The polynucleotide encoding human FcγRIIIa of the obtained plasmid and the region around the polynucleotide were subjected to a cycle sequencing reaction using BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by Life Technologies) based on the chain terminator method. The nucleotide sequence was analyzed using an automated DNA sequencer Applied Biosystems 3130 Genetic Analyzer (manufactured by Life Technologies). In this analysis, an oligonucleotide consisting of the sequence of SEQ ID NO: 5 (5'-TAATACGACTCACTATAGGGG-3 ') or SEQ ID NO: 7 (5'-TATGCTAGTTATTGCTCAG-3') was used as a primer for sequencing. As a result of sequence analysis, a transformant expressing Fc-binding protein (SEQ ID NO: 9) in which Val176 of FcR9 was replaced with Phe was obtained.

Example 2 Preparation of Fc-binding protein (FcR9_F_Cys) to which a cysteine tag is added (1) An expression vector containing the polynucleotide of SEQ ID NO: 10 encoding the amino acid sequence of SEQ ID NO: 9 prepared in Example 1 PCR was performed using pET-FcR9_F as a template. The primers used in the PCR were SEQ ID NO: 11 (5'-TAGCCATGGGCCATGCGTACCGAAGATCTGCCGAAAGC-3 ') and SEQ ID NO: 12 (5'-CCCAAGCTTATCCCGCAGGTATCGTTGCGGCACCCTTGGG. In PCR, after preparing a reaction solution having the composition shown in Table 3, the reaction solution was heat-treated at 98 ° C. for 5 minutes, a first step at 98 ° C. for 10 seconds, a second step at 55 ° C. for 5 seconds, and a reaction at 72 ° C. The reaction was performed by repeating the reaction in which the third step of one minute was defined as one cycle was repeated 30 times.

(2) The polynucleotide obtained in (1) is purified, digested with restriction enzymes NcoI and HindIII, and then digested with restriction enzymes NcoI and HindIII in advance, and the expression vector pTrc-PelBV3 prepared by the method described in WO2015 / 199154. And the ligation product was used to transform E. coli W3110 strain.

(3) The resulting transformant was cultured in an LB medium containing 100 μg / mL carbenicillin, and the expression vector pTrc-FcR9_F_Cys was obtained using QIAprep \ Spin \ Miniprep \ kit (manufactured by Qiagen).

(4) Analysis of the nucleotide sequence of pTrc-FcR9_F_Cys was performed by sequencing an oligonucleotide comprising the sequence described in SEQ ID NO: 13 (5′-TGTGGTATGGCTGTGCAGGG-3 ′) or SEQ ID NO: 14 (5′-TCGGCATGGGGTCAGGGTG-3 ′). , Except that it was used in Example 1.アミノ酸 The amino acid sequence of the polypeptide expressed by the expression vector pTrc-FcR9_F_Cys is shown in SEQ ID NO: 15, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 16. In SEQ ID NO: 15, the region from the 1st methionine (Met) to the 22nd alanine (Ala) is an improved PelB signal peptide, and the region from the 24th glycine (Gly) to the 199th glutamine (Gln) has Fc binding property. The amino acid sequence of the protein (corresponding to the region from position 17 to position 192 of SEQ ID NO: 1), and the glycine (Gly) at position 200 to glycine (Gly) at position 207 are cysteine tag sequences.

Example 3 Preparation of FcR9_F_Cys (1) 400 mL of 2YT liquid medium (peptone 16 g / L, yeast extract, containing 100 μg / mL carbenicillin in a 2 L baffle flask containing the transformant expressing FcR9_F_Cys prepared in Example 2) (10 g / L, 5 g / L sodium chloride), and precultured by aerobically shaking at 37 ° C. overnight.

(2) Glucose 10 g / L, yeast extract 20 g / L, trisodium phosphate dodecahydrate 3 g / L, disodium hydrogen phosphate dodecahydrate 9 g / L, ammonium chloride 1 g / L and carbenicillin 100 mg / L 180 mL of the culture solution of (1) was inoculated into 1.8 L of a liquid medium containing L, and main culture was performed using a 3 L fermenter (manufactured by Biot). Main culture was started under the conditions of a temperature of 30 ° C., a pH of 6.9 to 7.1, an aeration of 1 VVM, and a dissolved oxygen concentration of 30% saturation. To control the pH, 50% phosphoric acid was used as the acid and 14% ammonia water was used as the alkali, and the dissolved oxygen was controlled by changing the stirring speed, and the stirring rotation speed was set to a lower limit of 500 rpm and an upper limit of 1000 rpm. . After the start of the culture, when the glucose concentration cannot be measured, the feed medium (glucose 248.9 g / L, yeast extract 83.3 g / L, magnesium sulfate heptahydrate 7.2 g / L) is dissolved in dissolved oxygen (DO). ).

(3) When the absorbance at 600 nm (OD600 nm) reaches about 150, the culture temperature is lowered to 25 ° C., and after confirming that the set temperature has been reached, the final concentration is adjusted to 0.5 mM. IPTG was added, and the culture was continued at 25 ° C.

(4) After about 48 hours from the start of the culture, the culture was stopped, and the culture was centrifuged at 8,000 rpm at 4 ° C. for 20 minutes to collect the cells.

(5) The collected cells were suspended in 20 mM Tris-HCl buffer (pH 7.0) at a concentration of 5 mL / 1 g (cells), and an ultrasonic generator (Insonator 201M (trade name), manufactured by Kubota Corporation) ) Was disrupted at 4 ° C. for about 10 minutes at an output of about 150 W. The cell lysate was centrifuged twice at 8000 rpm for 20 minutes at 4 ° C., and the supernatant was recovered.

(6) The supernatant obtained in (5) was previously equilibrated with 20 mM phosphate buffer (8 mM sodium dihydrogen phosphate, 12 mM disodium hydrogen phosphate) (pH 7.0) to 140 mL of TOYOPEARL @ CM- A VL32 × 250 column (manufactured by Merck Millipore) packed with 650M (manufactured by Tosoh Corporation) was applied at a flow rate of 5 mL / min. After washing with the buffer used for equilibration, elution was carried out with a 20 mM phosphate buffer (pH 7.0) containing 0.5 M sodium chloride.

(7) The eluate obtained in (6) was packed with 90 mL of IgG Sepharose (manufactured by GE Healthcare) previously equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride. 20 columns (manufactured by GE Healthcare) were applied. After washing with the buffer used for equilibration, elution was performed with 0.1 M glycine hydrochloride buffer (pH 3.0). The pH of the eluate was returned to around neutral by adding 1/4 of the amount of the eluate to 1 M Tris-HCl buffer (pH 8.0).

約 About 20 mg of high-purity FcR9_F_Cys was obtained by the purification.

Example 4 Preparation of Fc-Binding Protein (FcR9_F) -Immobilized Gel and Separation of Antibodies (1) Hydroxyl groups on the surface of 2 mL of hydrophilic vinyl polymer for separating agent (manufactured by Tosoh: packing material for liquid chromatography) were converted to iodoacetyl groups. After activating with FcR9_F_Cys prepared in Example 3, 4 mg was reacted to obtain an FcR9_F-immobilized gel.

(2) An FcR9_F column was prepared by packing 1.2 mL of the FcR9_F-immobilized gel prepared in (1) into a φ4.6 mm × 75 mm stainless steel column.

(3) The FcR9_F column prepared in (2) was connected to a high performance liquid chromatography apparatus (manufactured by Tosoh Corporation), and equilibrated with an equilibration buffer of 20 mM acetate buffer (pH 5.5) containing 50 mM sodium chloride. .

(4) 5 μL of a monoclonal antibody (Rituxan, manufactured by Zenyaku Kogyo, mouse and human chimeric antibody) diluted to 1.0 mg / mL with PBS (Phosphate Buffered Saline) (pH 7.4) at a flow rate of 0.6 mL / min. Was added.

(5) After washing with an equilibration buffer solution at a flow rate of 0.6 mL / min for 2 minutes, a pH gradient with 10 mM glycine hydrochloride buffer (pH 3.0) (10 mM glycine hydrochloride buffer (pH 3.0) in 28 minutes) The adsorbed monoclonal antibody was eluted with a gradient of 100%.

The results (elution pattern) are shown in FIG. Because the monoclonal antibody interacts with the Fc binding protein, it was separated into multiple peaks instead of a single peak as in gel filtration chromatography. The first peak with the earlier elution time was designated as peak A, and the third peak with the later elution time was designated as peak B.

Example 5 Separation of human-derived antibody using Fc-binding protein (FcR9_F) -immobilized gel The same operation as in Example 4 was carried out except that a human-derived gamma globulin preparation (manufactured by Chemistry and Serological Therapy Laboratory) was used as the antibody. FIG. 2 shows the separation pattern of the human-derived gamma globulin preparation. A separation pattern different from that of the monoclonal antibody (Rituxan) of Example 4 (FIG. 1) was obtained.

In addition, the human-derived antibody contained in each fraction was collected by repeatedly collecting peaks C and D in FIG. 2, which are characteristic separation peaks of human-derived antibodies.

Example 6 Analysis of Sugar Chain Structure of Monoclonal Antibody Structural analysis of the sugar chain of the antibody contained in the peak A and peak B fractions separated in Example 4 was carried out in the same manner as the method described in JP-A-2016-169197. It was carried out in. The results are shown in FIG. Man represents mannose, GlcNAc represents N-acetylglucosamine, Gal represents galactose, Fuc represents fucose, and NeuAc represents N-acetylneuraminic acid.

Example 7 Analysis of Sugar Chain Structure of Human-Derived Antibody Structural analysis of the sugar chain of the antibody contained in the fraction of peak C and peak D fractioned in Example 5 was performed in the same manner as in Example 6, The results of the analysis of the sugar chain structure of the antibody contained in the fraction of peak D are shown in FIG.

From the results of Example 6 and Example 7, it was found that the presence or absence of galactose contributed to the separation of the antibody by using the FcR column packed with the gel on which the Fc binding protein was immobilized (see Table 3 for G0F). Comparison of the composition ratio of A and B, and similarly comparison of G1F and G2F. The number between the abbreviations G and F indicates the number of galactose.) Similarly, it is clear that antibodies are separated by the presence or absence of sialic acid. It became. Furthermore, comparison of FIG. 3 and FIG. 4 revealed that human-derived gamma globulin has a human-specific sugar chain structure unlike Rituxan, a mouse chimeric antibody that is a commercially available antibody drug. Among these human-specific sugar chain structures, an antibody having a sugar chain structure to which sialic acid is added can serve as an index of a specific disease, and therefore, by using the method of the present invention, simple measurement of disease can be achieved. In addition, early detection of abnormalities by comparison with healthy persons, prognostic management of affected patients, and the like can be performed.

Example 8 Separation of human-derived antibodies of different ages using Fc-binding protein (FcR9_F) -immobilized gel (1) Blood was collected from a healthy individual who obtained informed consent. The age and gender of healthy subjects are shown below.
(A sample) 36 years old, woman (B sample) 44 years old, woman (C sample) 55 years old, woman

(2) Purification of gamma globulin from serum obtained by centrifuging the blood collected in (1) using a gamma globulin G purification kit (manufactured by Thermo @ Fisher @ Scientific) having Protein G immobilized on a solid phase. did.

(3) Using the gamma globulin obtained in (2) as an elution condition, wash with an equilibration buffer at a flow rate of 0.6 mL / min for 5 minutes, and then use a 10 mM glycine hydrochloride buffer (pH 3.0). A gamma globulin separation pattern was obtained in the same manner as in Example 4, except that the adsorbed gamma globulin was eluted with a pH gradient (a gradient in which 10 mM glycine hydrochloride buffer (pH 3.0) becomes 100% in 30 minutes). Was.

(4) The separation pattern obtained in (3) is defined as a first peak, a second peak, a third peak, and a fourth peak in the order of the elution time which is shortest after the application of the pH gradient, and the third peak is defined. Was normalized with the detected value of 1 as 1.

結果 The results of Example 8 are shown in FIG. It can be seen that as the age increases in the order of the B sample and the C sample as compared with the A sample, the proportion of gamma globulin which is eluted earlier increases. In particular, it can be seen that the peak area% of the first peak and the second peak is increased in the sample C as compared with the sample A and the sample B. The fact that the proportion of gamma globulin having a long elution time is large means that the gamma globulin has a high binding ability to natural killer cells, monocytes and macrophages, and can promote the activation of the cells. On the other hand, when the proportion of gamma globulin having a short elution time is large, activation of the cells cannot be sufficiently obtained, and the risk of developing a disease affected by the activity of the cells increases. Examples of such diseases include infectious diseases caused by viruses and bacteria, cancers, allergies, and inflammatory diseases. The fluctuation of the gamma globulin separation pattern in Example 8 shows that the risk of onset increases with an increase in age.

Example 9 Separation of human-derived antibodies of different ages using an Fc-binding protein-immobilized gel (1) Blood was collected from a healthy subject who obtained informed consent. The age group and the number of samples of healthy subjects are shown below.
18-29: 23 specimens 30-39: 21 specimens 40-49: 21 specimens 50-59: 24 specimens 60-75: 15 specimens

(2) Serum obtained by centrifuging the blood collected in (1) was diluted 10-fold with PBS, and then passed through a filter having a diameter of 0.2 μm (Merck @ Millipore) to prepare a measurement sample.

(3) A gamma globulin separation pattern was obtained in the same manner as in Example 8 (3) except that 10 μL of the measurement sample obtained in (2) was added at a flow rate of 0.6 mL / min.

(4) The separation pattern obtained in (3) is defined as a first peak, a second peak, a third peak, and a fourth peak in order of the elution time which is shortest after the application of the pH gradient, and the first peak is defined. The value obtained by dividing the area value of by the total area value from the start of the application of the pH gradient to the end of the application of the pH gradient was defined as the first peak area%.

結果 The results of Example 9 are shown in FIG. Gamma globulin eluted earlier because the value of the first peak area% significantly increased as the age increased in the order of the sample in the 50's and the sample in the age of 60 or more as compared with the sample under the age of 50 It can be seen that the ratio of increases. Also, it can be seen that, even in a sample younger than 50 years old, a sample having a high value of the first peak area% exists at a low ratio. This result indicates that the immune activity decreases with aging when evaluated as a population, and indicates that the immune activity can be reduced even in young people when evaluated as an individual.

Example 10 Isolation of gamma globulin from cancer patients Gamma globulin was isolated in the same manner as in Example 9, except that blood was collected from healthy subjects who obtained informed consent and pancreatic cancer, gastric cancer, and breast cancer patients. Then, the first peak area% was determined.

Example 11 Separation of gamma globulin from a patient with an autoimmune disease Gamma globulin was separated by the same method as in Example 9 except that rheumatism from which informed consent was obtained and blood collected from a patient with Sjogren's syndrome were used. The area% was determined.

結果 The results of Examples 10 and 11 are shown in FIGS. Compared with a sample of a healthy subject, pancreatic cancer (panel a in FIG. 7), stomach cancer (panel b in FIG. 7), breast cancer (panel c in FIG. 7), rheumatism (panel a in FIG. 8), Sjogren's syndrome ( In the patient of FIG. 8 panel b), the value of the first peak area% was significantly increased. Further, it was found that the value of the first peak area% increased depending on the stage of the cancer disease. The term “stage” is based on the stage classification defined by the International Union for Cancer Control (UICC). This result indicates that the immune activity related to the Fc-binding protein is reduced in patients with cancer diseases and autoimmune diseases, and the first peak area% is significantly increased especially in cancer diseases. This indicates that the immune activity is greatly reduced. In cancer diseases or autoimmune diseases, it is considered that the decrease in the immune activity is a factor causing the onset or the onset decreases the immune activity. Since the value of the first peak area% of the healthy subject and the specimen is different between the disease specimen, it can be used for diagnosis of the disease, and the value of the first peak area% depending on the stage of the cancer disease. Can be used to evaluate the progression and malignancy of cancer.

Example 12 Evaluation of Gamma Globulin Separation Derived from Cancer Patients Corrected by Age (1) A method similar to that of Example 9 except that blood collected from renal and colon cancer patients who obtained informed consent was used. The gamma globulin was separated by the above, and the first peak area% was determined.

(2) A correlation curve between the first peak area% obtained in the measurement of the healthy subject sample of Example 9 and age is determined by polynomial approximation, and each age of the sample is introduced into the equation of the correlation curve. The value was calculated as a correction value.

(3) Corrected first peak area% by subtracting the value calculated from (2) from the healthy subject of Example 9, the pancreatic cancer of Example 10, and the first peak area% determined by (1). I asked.

結果 The results of Example 12 are shown in FIG. Comparing a healthy subject's sample with pancreatic, renal, and colon cancer based on the corrected first peak area% obtained by correcting the first peak area% that increases with aging, The corrected first peak area% significantly increased as compared with the control. The corrected first peak was similar to the significant increase in the first peak area% found in Example 10 in which a healthy subject sample was compared with pancreatic cancer based on the uncorrected first peak area%. Since the increase was also confirmed in the area%, it can be seen that the difference in the IgG separation pattern between the healthy subject and the cancer patient can be confirmed even when the correction considering the age is performed.

Example 13 Evaluation of Gamma Globulin Separation Derived from Pancreatic Cancer and Pancreatitis Patients (1) Except that pancreatic cancer from which informed consent was obtained and blood collected from pancreatitis patients were used, Examples 9 (2) and (3) Gamma globulin was separated in the same manner as described above.

(2) The separation pattern obtained in (1) is defined as a first peak, a second peak, and a third peak in order of a peak having a shorter elution time after the application of a pH gradient, and the area value of the first peak is defined as The value obtained by dividing by the total area value from the start to the end of the application of the pH gradient was defined as the first peak area%, and the corrected first peak area was corrected in the same manner as in Examples 12 (2) and (3). % And the area of the third peak was also determined.

結果 The results of Example 13 are shown in FIG. In panel (a) of FIG. 10, the value of the corrected first peak area% was significantly reduced in patients with pancreatitis as compared with the pancreatic cancer patient sample. In addition, when compared with the healthy subject sample of Example 12, the corrected first peak area% of the patient with pancreatitis has almost the same value, and it can be seen that the value of the corrected first peak area% does not change only when the healthy person has pancreatitis. . When the discrimination between pancreatic cancer and pancreatitis was evaluated by the AUC value based on the ROC curve using the corrected first peak area%, it was 0.83. On the other hand, in the panel (b) of FIG. 10, when compared with the third peak area, the value was significantly increased in pancreatitis patients as compared with pancreatic cancer patient samples. When the discriminability between pancreatic cancer and pancreatitis is evaluated by the AUC value based on the ROC curve at the third peak area, it is 1.00. It is possible to determine whether or not it is accurate.

Example 14 Gamma Globulin Separation Evaluation from Healthy Smoking / Non-smoking Persons Gamma globulin was separated in the same manner as in Example 12 except that blood collected from healthy persons who obtained informed consent from smoking and non-smoking were used. Then, the corrected first peak area% was determined.

結果 The results of Example 14 are shown in FIG. The value of the corrected first peak area% was significantly increased in the healthy person who smoked compared to the healthy person who did not smoke. The increase in the value of the corrected first peak area% is the same as the tendency detected from the cancer patient from Example 12, and it can be seen that smoking increases the risk of cancer.

Example 15 Separation of human-derived antibodies of different ages using different Fc binding protein (FcR9_F or FcR9_V) immobilized gel (1) The amino acid sequence of the expressed polypeptide is represented by SEQ ID NO: 17, and the polypeptide encoding the polypeptide is represented by SEQ ID NO: 17. A cysteine-tagged Fc-binding protein (FcR9_V_Cys) was prepared in the same manner as in Example 3, except that the expression vector whose nucleotide sequence was shown in SEQ ID NO: 18 was used.

(2) An FcR9_V column was prepared in the same manner as in Examples 4 (1) and (2) except that the Fc-binding protein prepared in (1) was used.

(3) Blood was collected from healthy subjects who obtained informed consent. The age and gender of healthy subjects are shown below.
(Healthy person A) 21 years old, male (healthy person B) 26 years old, male (healthy person C) 36 years old, male (healthy person D) 47 years old, male

(4) A gamma globulin separation pattern was obtained in the same manner as in Examples 8 (2) and (3) except that an FcR9_F column or FcR9_V column was used as the column.

(5) The separation pattern obtained in (4) is defined as a first peak, a second peak, and a third peak in the order of elution time after the start of the application of the pH gradient, and the area value of the first peak is defined as The value obtained by dividing by the total area value from the start of the application of the pH gradient to the end thereof was defined as the first peak area%. Furthermore, the value obtained by dividing the height of the first peak by the total value of the heights of the respective peaks was defined as the first peak height%.

結果 The results of Example 15 are shown in FIG. In panel (a) of FIG. 12, it is a figure which shows the measured value of a healthy subject sample measured with the FcR9_F column or the FcR9_V column. From this figure, it can be seen that the value of the first peak height% increases with aging, and further, using a column packed with an insoluble carrier on which two types of Fc-binding proteins having different amino acid sequences are immobilized. Also, the same tendency that the first peak height% increased with aging was obtained. This result indicates that it is possible to detect the disease, the risk of developing the disease and / or the degree of progress of the disease, and the degree of progress of aging if the Fc-binding protein is not limited to the amino acid sequence. . Further, in the panel (b) of FIG. 12, the first peak area% and the first peak height% are shown as the measured values of the healthy subject measured by the FcR9_V column, respectively. Comparing the area% and the height% respectively, it can be seen that the value increases with aging in both cases, and that it is possible to evaluate accurately using either value.

According to the present invention, antibodies can be separated based on the difference in sugar chain structure. According to one embodiment of the present invention, the presence or absence of a disease in a subject, the risk of developing a disease, the degree of progression of a disease, and / or the degree of progression of aging can be detected using the characteristics of an antibody separation pattern as an index. .

Claims

The presence or absence of the disease, the risk of developing the disease, the degree of progress of the disease, and / or a method for detecting the degree of progress of aging,
The following step (c):
(C) detecting the presence or absence of a disease in the subject, the risk of developing the disease, the degree of progression of the disease, and / or the degree of progression of aging in the subject using the data relating to the antibody separation pattern as an index,
The data is characteristic of the separation pattern of the antibody,
The method wherein the data is obtained by the following steps (a) and (b):
(A) adding a solution containing an antibody obtained from the subject to a column packed with an insoluble carrier having Fc-binding protein immobilized thereon, and adsorbing the antibody to the carrier;
(B) a step of eluting the antibody adsorbed on the carrier using an eluent to obtain the data.
方法 The method according to claim 1, comprising the steps (a) and (b) before the step (c).
3. The method according to claim 1, further comprising a step of equilibrating the column by adding an equilibration liquid to the column before the step (a).
The method according to any one of claims 1 to 3, wherein the acquisition of the data includes a step of obtaining a separation pattern of the antibody, and a step of extracting the feature from the separation pattern.
The method according to any one of claims 1 to 4, wherein the characteristic is a peak area and / or a peak height.
The method according to any one of claims 1 to 5, wherein the characteristic is peak area% and / or peak height%.
The method according to any one of claims 1 to 6, wherein the characteristic is a characteristic of one or more peaks selected from a first peak, a second peak, and a third peak.
The method according to any one of claims 1 to 7, wherein the characteristic is a characteristic of a first peak.
The method according to any one of claims 1 to 8, wherein the step (c) includes a step of comparing the data with data on an antibody separation pattern obtained from a control subject.
The method according to any one of claims 1 to 9, wherein the disease is one or more diseases selected from cancer, autoimmune disease, infectious disease, allergy, inflammatory disease, cachexia, and age-related disease. Item 2. The method according to item 1.
The disease according to any one of claims 1 to 10, wherein the disease is one or more diseases selected from pancreatic cancer, gastric cancer, breast cancer, colorectal cancer, kidney cancer, rheumatism, Sjogren's syndrome, and pancreatitis. The method described in the section.
The method according to any one of claims 1 to 11, wherein the Fc binding protein is a polypeptide according to any one of the following (1) to (4):
(1) It includes the 17th to 192nd amino acid residues of the amino acid sequence of SEQ ID NO: 1, provided that at least the 176th valine is substituted with phenylalanine in the 17th to 192nd amino acid residues. A polypeptide;
(2) It includes the 17th to 192nd amino acid residues of the amino acid sequence of SEQ ID NO: 1, provided that at least the 27th valine is the glutamic acid and the 29th amino acid is the 17th to the 192nd amino acid residues. Phenylalanine to isoleucine, 35th tyrosine to asparagine, 48th glutamine to arginine, 75th phenylalanine to leucine, 92nd asparagine to serine, 117th valine to glutamic acid, 121st glutamic acid Is a polypeptide in which is substituted with glycine, phenylalanine at position 171 with serine, and valine at position 176 with phenylalanine;
(3) It includes the 17th to 192nd amino acid residues of the amino acid sequence of SEQ ID NO: 1, provided that at least the 17th to 192nd amino acid residues have at least 27th valine in glutamic acid and 29th in glutamic acid. Phenylalanine to isoleucine, 35th tyrosine to asparagine, 48th glutamine to arginine, 75th phenylalanine to leucine, 92nd asparagine to serine, 117th valine to glutamic acid, 121st glutamic acid Has been replaced by glycine and phenylalanine at position 171 by serine;
(4) A polypeptide comprising the amino acid sequence of the polypeptide of any of (1) to (3) above, provided that the amino acid sequence has 1 to 10 amino acid mutations at positions other than the above substitutions.
A composition containing two or more antibodies, wherein the composition falls under two or more of the following I to IX:
I. A value obtained by dividing the content of the antibody having G1Fa by the content of the antibody having G0F is 0.4 or less by weight;
II. A value obtained by dividing the content of the antibody having G2F by the content of the antibody having G0F is 0.2 or less by weight;
III. The content of the antibody with G2F + 2SA divided by the content of the antibody with G0F is less than or equal to 0.03 by weight;
IV. The value obtained by dividing the content of the antibody having G1Fb by the content of the antibody having G1Fa is 0.5 or more by weight;
V. A value obtained by dividing the content of the antibody having G2F by the content of the antibody having G1Fb is 0.6 or less by weight;
VI. A value obtained by dividing the content of the antibody having G2F + SA by the content of the antibody having G1Fb is 0.3 or less by weight;
VII. The content of the antibody with G2F + 2SA divided by the content of the antibody with G1Fb is less than or equal to 0.12 by weight;
VIII. The ratio of the content of the antibody having G2 + SA to the total content of the antibody is 0.2% or less by weight;
IX. The ratio of the content of the antibody having G2 + 2SA to the total content of the antibody is 0.2% or less by weight.
A composition containing two or more antibodies, wherein the composition falls under two or more of the following I to IX:
I. A value obtained by dividing the content of the antibody having G1Fa by the content of the antibody having G0F is 1.8 or more by weight;
II. A value obtained by dividing the content of the antibody having G2F by the content of the antibody having G0F is 0.6 or more by weight;
III. The content of the antibody with G2F + 2SA divided by the content of the antibody with G0F is greater than or equal to 0.06 by weight;
IV. A value obtained by dividing the content of the antibody having G1Fb by the content of the antibody having G1Fa is 0.3 or less by weight;
V. The content of the antibody with G2F divided by the content of the antibody with G1Fb is greater than or equal to 3.0 by weight;
VI. A value obtained by dividing the content of the antibody having G2F + SA by the content of the antibody having G1Fb is 0.6 or more by weight;
VII. The content of the antibody with G2F + 2SA divided by the content of the antibody with G1Fb is greater than or equal to 0.3 by weight;
VIII. The ratio of the content of the antibody having G2 + SA to the total content of the antibody is 2% or more by weight;
IX. The ratio of the content of the antibody having G2 + 2SA to the total content of the antibodies is 0.6% or more by weight.